Aptamer protecting material and biosensor

Abstract

An analyte monitoring sensor configured for in vivo measurement of at least one analyte is provided, the sensor comprising: a substrate having a substrate surface; an aptamer protection layer encapsulating at least a portion of the substrate surface, the aptamer protection layer being permeable to the at least one analyte; and one or more aptamer conjugates associated with at least a portion of the substrate surface and positioned between the aptamer protection layer and the substrate to obtain a measurement related to the at least one analyte in vivo. Methods of extending the in vivo performance of the analyte monitoring sensor are also described.

Description

[Technical Field]

[0001] The present disclosure is directed to protective materials for aptamer-based biosensor constructs or devices suitable for wholly or partially implantation in a subject for continuous monitoring of an analyte. [Background technology]

[0002] Aptamer biosensors (ABs) are a class of affinity biosensors in which the recognition element is an aptamer (single-stranded DNA / RNA) with specific affinity for the analyte, and such aptamer-analyte interaction induces a measurable transduction signal (optical or electrical). Existing aptamer biosensors (ABs) are limited by their observed poor stability when placed in physiologically relevant environments, which is due, at least in part, to the detachment of the aptamer monolayer from the substrate surface or the detachment of the bottom monolayer used to immobilize the aptamer. These detachment events limit the application of ABs for continuous analyte monitoring in physiological environments. Another weakness of AB sensors, especially electrochemical aptamer biosensors (EABs), is that their bioelectronic interface degrades upon continuous electrochemical interrogation and / or biofouling; this process is typically seen as a faradaic degradation and an increase in charging current over time. This progressive degradation limits the operational lifetime of EABs in vivo to 12 hours or less, a period much shorter than the elimination half-life of most drugs in humans. Summary of the Invention

[0003] In a first example, an analyte monitoring sensor configured for in vivo measurement of at least one analyte is provided, the sensor comprising: a substrate having a substrate surface; an aptamer protection layer encapsulating at least a portion of the substrate surface, the aptamer protection layer being permeable to at least one analyte; one or more aptamer conjugates associated with at least a portion of the substrate surface and positioned between the aptamer protection layer and the substrate to obtain a measurement related to the at least one analyte in vivo; and a reversible redox moiety bound to the one or more aptamer conjugates.

[0004] In one embodiment, alone or in combination with any one of the preceding embodiments, at least a portion of the substrate is a conductive metal. In one embodiment, alone or in combination with any one of the preceding embodiments, at least a portion of the substrate is gold, carbon, graphene, or graphene oxide. In one embodiment, alone or in combination with any one of the preceding embodiments, at least a portion of the substrate comprises pores having an average pore size of nanometer and / or micrometer dimensions.

[0005] In one embodiment, alone or in combination with any one of the preceding embodiments, at least a portion of the substrate surface further comprises one or more co-adsorbents. In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more co-adsorbents independently comprise multiple functional groups.

[0006] In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more co-adsorbents independently provide one or more of a surface energy range, a phase separation range, and an intermolecular interaction range between the one or more aptamers and the aptamer-protecting layer. In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more co-adsorbents independently provide an ionic strength, a pH range, or a pH buffer, and the one or more co-adsorbents are present in an amount capable of adjusting or maintaining the ionic strength, pH range, or pH buffer in proximity to the at least one aptamer conjugate.

[0007] In one embodiment, alone or in combination with any one of the preceding embodiments, at least a portion of the substrate surface, the one or more co-adsorbents, and a portion of the remaining portion of the substrate surface comprise one or more aptamer conjugates. In one embodiment, alone or in combination with any one of the preceding embodiments, at least a portion of the substrate surface comprises one or more co-adsorbents, and a portion of the remaining portion of the substrate surface comprises one or more aptamer conjugates physically or chemically bound thereto. In one embodiment, alone or in combination with any one of the preceding embodiments, one or more co-adsorbents are physically or chemically bound to the substrate surface, and a portion of the remaining portion of the substrate surface comprises one or more aptamer conjugates physically or chemically bound thereto.

[0008] In one embodiment, alone or in combination with any one of the preceding embodiments, the co-adsorbate comprises a self-assembled monolayer (SAM). In one embodiment, alone or in combination with any one of the preceding embodiments, the co-adsorbate bound or tethered to the substrate is represented as follows:

[0009] [ka] In the formula, X is -OH, -NHR1, -NH2, or -SH; R1 is acyclic alkyl, substituted or unsubstituted cyclic alkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heteroalkyl, or substituted or unsubstituted heterocyclic; and a is 1 to 3.

[0010] In one embodiment, alone or in combination with any one of the preceding embodiments, the co-adsorbent comprises a mono- or polyfunctional alkanethiol, hydroxyalkyl mercaptan, alkoxy mercaptan, alkylaryl thiol, hydroxyalkylaryl thiol, hydroxyalkylaryl mercaptan, alkylaryl mercaptoalkanol, alkyl mercaptophenol, alkyl mercaptocatechol, aryl mercaptophenol, aryl mercaptocatechol, alkoxyaryl thiol, alkoxyaryl mercaptan (hereinafter collectively referred to as "thiol co-adsorbent"). In one embodiment, alone or in combination with any one of the preceding embodiments, the thiol functional group of the mono- or polyfunctional alkanethiol is covalently bonded to at least a portion of the substrate surface.

[0011] In one embodiment, alone or in combination with any one of the preceding embodiments, a thiol co-adsorbent is covalently bonded to the gold substrate surface. In one embodiment, alone or in combination with any one of the preceding embodiments, the co-adsorbent comprises a mono- or poly-functional mercaptoalkanol, a benzylmercaptoalkanol, or an arylmercaptoalkanol (hereinafter collectively referred to as "(aryl)mercaptoalkanol"). In one embodiment, alone or in combination with any one of the preceding embodiments, the thiol functional group of the mono- or poly-functional (aryl)mercaptoalkanol is covalently bonded to at least a portion of the substrate surface. In one embodiment, alone or in combination with any one of the preceding embodiments, the thiol functional group of the mono- or poly-functional (aryl)mercaptoalkanol is covalently bonded to at least a portion of the gold substrate surface.

[0012] In one aspect, alone or in combination with any one of the preceding aspects, at least a portion of the substrate surface comprises zwitterionic repeat groups. In one aspect, alone or in combination with any one of the preceding aspects, the zwitterionic repeat groups comprise betaine groups. In one aspect, alone or in combination with any one of the preceding aspects, the zwitterionic repeat groups comprise ammoniophosphate or a lecithin analog.

[0013] In one embodiment, alone or in combination with any one of the preceding embodiments, the zwitterionic repeat group comprises an ammoniophosphonate. In one embodiment, alone or in combination with any one of the preceding embodiments, the zwitterionic repeat group comprises an ammoniophosphinate. In one embodiment, alone or in combination with any one of the preceding embodiments, the zwitterionic repeat group comprises an ammoniosulfonate. In one embodiment, alone or in combination with any one of the preceding embodiments, the zwitterionic repeat group comprises an ammoniosulfate. In one embodiment, alone or in combination with any one of the preceding embodiments, the zwitterionic repeat group comprises an ammoniocarboxylate.

[0014] In one embodiment, alone or in combination with any one of the preceding embodiments, the zwitterionic repeating group comprises an alkanethiol betaine, a phenylthiol betaine, or a benzylthiol betaine. In one embodiment, alone or in combination with any one of the preceding embodiments, the alkanethiol, phenylthiol, or benzylthiol is linear and comprises multiple betaine groups along its chain. In one embodiment, alone or in combination with any one of the preceding embodiments, the alkanethiol, phenylthiol, or benzylthiol is a terminally terminated mono- or dithiol having at least one betaine group along its chain or aromatic ring. In one embodiment, alone or in combination with any one of the preceding embodiments, the zwitterionic repeating group comprises an n-mercaptoalkanol betaine. In one embodiment, alone or in combination with any one of the preceding embodiments, the mercaptoalkanol is linear and comprises multiple betaine groups along its chain. In one embodiment, alone or in combination with any one of the preceding embodiments, the mercaptophenol comprises one or more betaine groups attached to an aromatic ring. In one embodiment, alone or in combination with any one of the preceding embodiments, the mercaptoalkanol or mercaptophenol is a 1,2-dithiol, 1,3-dithiol, or 1,4-dithiol of an alkyl or aromatic hydrocarbon compound.

[0015] In one aspect, alone or in combination with any one of the preceding aspects, the thiol group of the terminally terminated dithiolalkanethiol is covalently bonded to the substrate surface. In one aspect, alone or in combination with any one of the preceding aspects, the thiol group of the mercaptoalkanol is covalently bonded to the substrate surface.

[0016] In one embodiment, alone or in combination with any one of the previous embodiments, the co-adsorbent has the following structure:

[0017] [ka] (In the formula,

[0018] [ka] represents a hydrocarbon chain; R1 and R2 are independently branched or unbranched acyclic alkyl, substituted or unsubstituted cyclic alkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocyclic; X is -OH, -NHR1, -NH2, or -SH; and n is an integer from 2 to about 1000; or

[0019] [ka] wherein X is -OH, -NHR, -NH, or -SH; W, Y, and Z are independently branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, any of which may be optionally substituted with O, OH, halogen, amido, or alkoxyl; R is H, branched or unbranched acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; and R, R, and R are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl. In one embodiment, one or more of R1, R2, R3, R4, R5, and Z are covalently or ionically bonded to the APL.

[0020] In one embodiment, alone or in combination with any one of the preceding embodiments, at least a portion of the substrate surface comprises a covalently bound aliphatic amine. In one embodiment, alone or in combination with any one of the preceding embodiments, at least a portion of the substrate surface comprises a covalently bound aminoalkanoic acid.

[0021] In one aspect, alone or in combination with any one of the preceding aspects, at least a portion of the carbon, graphene, or graphene oxide substrate surface comprises a covalently bound aminoalkanoic acid. In one aspect, alone or in combination with any one of the preceding aspects, at least a portion of the carbon, graphene, or graphene oxide substrate surface comprises a covalently bound aminoalkanoic acid, which is also covalently bound to one or more aptamer conjugates.

[0022] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer is at least partially crosslinked using an amount of a crosslinker. In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises a conductive polymer. In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises a zwitterionic group compound. In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises a zwitterionic repeat group compound.

[0023] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer provides an ionic strength, pH range, or pH buffer. In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer provides an amount of zwitterionic repeat group compound present that can adjust or maintain the ionic strength, pH range, or pH buffer. In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer provides a free volume that allows for reversible conformational change of one or more aptamer conjugates present therein, the free volume being sufficient to provide a signal in the presence of at least one analyte.

[0024] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises a functionalized polymer. In one embodiment, alone or in combination with any one of the preceding embodiments, the functionalized polymer comprises an alkanethiol group. In one embodiment, alone or in combination with any one of the preceding embodiments, the alkanethiol group is present at the end of the functionalized polymer chain. In one embodiment, alone or in combination with any one of the preceding embodiments, the alkanethiol group is present along the backbone of the functionalized polymer chain.

[0025] In one embodiment, alone or in combination with any one of the preceding embodiments, the functionalized polymer comprises a mercaptoalkanol group. In one embodiment, alone or in combination with any one of the preceding embodiments, the mercaptoalkanol group is present at the end of the functionalized polymer chain. In one embodiment, alone or in combination with any one of the preceding embodiments, the mercaptoalkanol group is present along the backbone of the functionalized polymer chain.

[0026] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises a functionalized polymer comprising one or more zwitterionic repeat groups. In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more zwitterionic repeat groups comprise a betaine compound or a derivative thereof. In one embodiment, alone or in combination with any one of the preceding embodiments, the zwitterionic repeat groups are present at the ends of the functionalized polymer chain. In one embodiment, alone or in combination with any one of the preceding embodiments, the zwitterionic repeat groups are present along the backbone of the functionalized polymer chain.

[0027] In one aspect, alone or in combination with any one of the preceding aspects, the functionalized polymer comprises an alkanethiol group and a zwitterionic repeating group. In one aspect, alone or in combination with any one of the preceding aspects, the functionalized polymer comprises an alkanethiol group and a betaine group. In one aspect, alone or in combination with any one of the preceding aspects, the functionalized polymer comprises a mercaptoalkanol group and a zwitterionic repeating group.

[0028] In one embodiment, alone or in combination with any one of the previous embodiments, the functionalized polymer comprises a mercaptoalkanol group and a betaine group.

[0029] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer is physically or chemically bound to at least a portion of the substrate surface. In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer is physically or chemically bound to at least a portion of the substrate surface, one or more aptamer conjugates are physically or chemically bound to at least a portion of the substrate surface, and a substantial remainder of the substrate surface further comprises a physically or chemically bound co-adsorbate.

[0030] In one aspect, alone or in combination with any one of the preceding aspects, the aptamer protection layer comprises at least one polymer segment selected from the group consisting of polyurethane, polyurea, poly(urethane urea), epoxide, polyolefin, polysiloxane, polyamide, polystyrene, polyacrylate, polyether, polyvinylpyridine, polyvinylpyrrolidone, polyester, polycarbonate, and copolymers thereof.

[0031] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises a segmented multiblock polymer. In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises a segmented multiblock polyurethane polymer. In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises a segmented multiblock polyurethane urea polymer.

[0032] In one embodiment, alone or in combination with any one of the previous embodiments, the segmented multiblock polymer comprises a soft segment and a hard segment. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment is hydrophobic or hydrophilic. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment is hydrophobic and hydrophilic. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment comprises a hydrophobic polyol and a hydrophilic polyol.

[0033] In one aspect, alone or in combination with any one of the preceding aspects, the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof. In one aspect, alone or in combination with any one of the preceding aspects, the soft segment is one or more segments comprising polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, zwitterionic repeating group polymer, and blends or copolymers thereof.

[0034] In one embodiment, alone or in combination with any one of the previous embodiments, the hard segments comprise urethane or urea groups.

[0035] In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more aptamer conjugates are physically associated with a portion of the substrate surface. In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more aptamer conjugates are covalently associated with a portion of the substrate surface.

[0036] In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more aptamer conjugates comprise an RNA or DNA nucleotide sequence. In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more aptamer conjugates comprise at least one of a 2'-O-methyl modification of nucleotides, a disulfide bridge, a 3' cap with an inverted 2-deoxythymidine, a 3'-3'-thymidine linkage at the 3' end, a 2'-F modification, and a double-stranded section. In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more aptamer conjugates comprise an RNA or DNA sequence having a first linker moiety at the 5' end and a reversible redox moiety at the 3' end. In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more aptamer conjugates comprise an RNA or DNA sequence having a first linker moiety at the 3' end and a reversible redox moiety at the 5' end.

[0037] In one embodiment, alone or in combination with any one of the preceding embodiments, the first linker moiety on the 5'-end or 3'-end of the aptamer comprises an amino group or a carboxyl group. In one embodiment, alone or in combination with any one of the preceding embodiments, the first linker moiety is physically or chemically bound to the substrate at the 5'-end. In one embodiment, alone or in combination with any one of the preceding embodiments, the first linker moiety is physically or chemically bound to the co-adsorbent at the 5'-end. In one embodiment, alone or in combination with any one of the preceding embodiments, the first linker moiety is physically or chemically bound to the substrate at the 3'-end. In one embodiment, alone or in combination with any one of the preceding embodiments, the first linker moiety is physically or chemically bound to the co-adsorbent at the 3'-end.

[0038] In one aspect, alone or in combination with any one of the preceding aspects, the one or more aptamer conjugates are glycopeptide antibiotic-binding aptamers. In one aspect, alone or in combination with any one of the preceding aspects, the one or more aptamer conjugates are vancomycin-binding aptamers. In one aspect, alone or in combination with any one of the preceding aspects, the one or more aptamer conjugates are neurotransmitter-binding aptamers. In one aspect, alone or in combination with any one of the preceding aspects, the one or more aptamer conjugates are dopamine- or glutamate-binding aptamers. In one aspect, alone or in combination with any one of the preceding aspects, the one or more aptamer conjugates are carbohydrate-, triglyceride-, or fatty acid-binding aptamers. In one aspect, alone or in combination with any one of the preceding aspects, the one or more aptamer conjugates are glucose-, glycerol-, or beta-hydroxybutyrate-binding aptamers.

[0039] In one embodiment, alone or in combination with any one of the preceding embodiments, one or more aptamer conjugates are physically or chemically bound to a self-assembled monolayer (SAM). In one embodiment, alone or in combination with any one of the preceding embodiments, one or more aptamer conjugates are physically or chemically bound to a mono- or polyfunctional alkanethiol or mercaptoalkanol. In one embodiment, alone or in combination with any one of the preceding embodiments, one or more aptamer conjugates are physically or chemically bound to an alkylthiolbetaine.

[0040] In one embodiment, alone or in combination with any one of the preceding embodiments, one or more aptamer conjugates are physically or chemically bound to an aliphatic amine. In one embodiment, alone or in combination with any one of the preceding embodiments, one or more aptamer conjugates are physically or chemically bound to an aminoalkanoic acid.

[0041] In one embodiment, alone or in combination with any one of the preceding embodiments, the reversible redox moiety comprises iron, iridium, ruthenium, osmium, a thiazine dye, or a derivative thereof. In one embodiment, alone or in combination with any one of the preceding embodiments, the reversible redox moiety comprises ferrocene or methylene blue.

[0042] In one aspect, alone or in combination with any one of the previous aspects, the sensor is configured for continuous, semi-continuous, sequential, or random signal acquisition. In one aspect, alone or in combination with any one of the previous aspects, the sensor further comprises one or more of a reference electrode, a working electrode, and a counter electrode. In one aspect, alone or in combination with any one of the previous aspects, the sensor further comprises one or more of a transmitter, a receiver, a controller, or a power source. In one aspect, alone or in combination with any one of the previous aspects, the sensor is configured for percutaneous insertion.

[0043] In a second example, a method for extending the end-of-life of an electrochemical aptamer biosensor (EAB) is provided, the method including: electrically associating at least one aptamer conjugate, the at least one aptamer conjugate comprising a reversible redox moiety, with a surface of a conductive substrate; encapsulating the at least one aptamer conjugate in an aptamer protection layer, the at least one aptamer conjugate being configured to undergo a reversible conformational change within the aptamer protection layer in response to interaction with an analyte to generate a detectable signal; controlling one or more of ionic strength, pH range, or pH buffering within the aptamer protection layer, surface phase separation of the aptamer protection layer, and intermolecular interactions between the at least one aptamer conjugate and the aptamer protection layer; and extending the end-of-life of the electrochemical aptamer sensor.

[0044] In one embodiment, alone or in combination with any one of the preceding embodiments, controlling ionic strength, providing a pH range, or pH buffering comprises incorporating one or more co-adsorbents into the aptamer protection layer, wherein the one or more co-adsorbents are present in an amount capable of adjusting or maintaining the ionic strength, pH range, or pH buffering.

[0045] In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more co-adsorbents comprise a zwitterionic betaine group. In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more co-adsorbents comprise a zwitterionic betaine group have the following structure:

[0046] [ka] (In the formula,

[0047] [ka] represents a hydrocarbon chain, and R1 and R2 are independently branched or unbranched acyclic alkyl, substituted or unsubstituted cyclic alkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocyclic, where X is -OH, -NHR1, -NH2, or -SH, and n is an integer from 2 to about 1000; or

[0048] [ka] wherein X is -OH, -NHR, -NH, or -SH; W, Y, and Z are independently branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, any of which may be optionally substituted with O, OH, halogen, amido, or alkoxyl; R is H, branched or unbranched acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; and R, R, and R are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl. In one embodiment, one or more of R1, R2, R3, R4, R5, and Z are covalently or ionically bonded to the APL.

[0049] In one embodiment, alone or in combination with any one of the preceding embodiments, the co-adsorbate is a terminally terminated dithiol having at least one betaine group along its chain. In one embodiment, alone or in combination with any one of the preceding embodiments, the thiol group of the terminally terminated dithiolalkanethiol is covalently bound to the substrate surface. In one embodiment, alone or in combination with any one of the preceding embodiments, the zwitterionic betaine group comprises a mercaptoalkanol betaine.

[0050] In one embodiment, alone or in combination with any one of the preceding embodiments, the mercaptoalkanol is linear and includes multiple betaine groups along the chain. In one embodiment, alone or in combination with any one of the preceding embodiments, the thiol group of the mercaptoalkanol is covalently bonded to the substrate surface.

[0051] In one embodiment, alone or in combination with any one of the preceding embodiments, controlling the ionic strength, pH range, or pH buffering comprises providing one or more zwitterionic betaine groups to the aptamer protection layer. In one embodiment, alone or in combination with any one of the preceding embodiments, controlling the ionic strength comprises providing a mercaptoalkanol and a zwitterionic betaine group to the aptamer protection layer. In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises an alkanethiol, a mercaptoalkanol, a benzylthiol, a mercaptophenol, and one or more zwitterionic groups.

[0052] In one embodiment, alone or in combination with any one of the preceding embodiments, controlling the ionic strength, pH range, or pH buffering comprises providing a pH adjusting composition or a pH buffering composition to the aptamer protection layer.

[0053] In one embodiment, alone or in combination with any one of the preceding embodiments, controlling the intermolecular interactions between the at least one aptamer conjugate and the aptamer-protection layer comprises providing a segmented multi-block polymer backbone to the aptamer-protection layer. In one embodiment, alone or in combination with any one of the preceding embodiments, the segmented multi-block polymer backbone comprises a polyurethane polymer. In one embodiment, alone or in combination with any one of the preceding embodiments, the segmented multi-block polymer backbone comprises a polyurethane-urea polymer.

[0054] In one embodiment, alone or in combination with any one of the previous embodiments, the segmented multiblock polymer comprises a soft segment and a hard segment. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment is hydrophobic or hydrophilic. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment is hydrophobic and hydrophilic. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment comprises a hydrophobic polyol and a hydrophilic polyol. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

[0055] In one aspect, alone or in combination with any one of the previous aspects, the soft segment is one or more segments comprising polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymer, and blends or copolymers thereof.

[0056] In one embodiment, alone or in combination with any one of the preceding embodiments, the segmented multiblock polymer comprises a soft segment and a hard segment. In one embodiment, alone or in combination with any one of the preceding embodiments, the hard segment comprises a urethane group or a urea group.

[0057] In one aspect, alone or in combination with any one of the preceding aspects, the soft segment is hydrophobic or hydrophilic. In one aspect, alone or in combination with any one of the preceding aspects, the soft segment is hydrophobic and hydrophilic. In one aspect, alone or in combination with any one of the preceding aspects, the soft segment comprises a hydrophobic polyol and a hydrophilic polyol. In one aspect, alone or in combination with any one of the preceding aspects, the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof. In one aspect, alone or in combination with any one of the preceding aspects, the soft segment is one or more segments comprising polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, zwitterionic repeating group polymer, and blends or copolymers thereof.

[0058] In one aspect, alone or in combination with any one of the previous aspects, reducing biofouling comprises providing an aptamer protection layer as defined in any one of the previous aspects.

[0059] In one aspect, alone or in combination with any one of the preceding aspects, reducing the separation of at least one aptamer from the surface of the conductive substrate comprises binding the at least one aptamer conjugate to the conductive substrate using carbodiimide binding to the conductive surface.

[0060] In one embodiment, alone or in combination with any one of the previous embodiments, reducing oxidation of the aptamer comprises incorporating one or more non-diffusible antioxidants into the aptamer protection layer.

[0061] In one embodiment, alone or in combination with any one of the previous embodiments, controlling the diffusion of at least one aptamer comprises at least partially crosslinking the aptamer protection layer.

[0062] In one aspect, alone or in combination with any one of the previous aspects, the end of life is extended by up to 1 day, 2 days, 1 week, 2 weeks, 3 weeks, or at least 1 month.

[0063] In another example, an aptamer protection layer configured for transdermal in vivo continuous online monitoring is provided, the aptamer protection layer comprising a functionalized polymer comprising at least one zwitterionic repeating group, a polymerizable zwitterionic monomer structure such as:

[0064] [ka] wherein X is O, NH, or NR4; Y and Z are independently acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, optionally substituted with OH, halogen, or alkoxyl; and R1, R3, R4, and R5 are independently H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; a functionalized polymer comprising an alkanethiol group; a functionalized polymer comprising an alkanethiol group and zwitterionic repeating groups; a functionalized polymer comprising an (aryl)mercaptoalkanol group; a functionalized polymer comprising an (aryl)mercaptoalkanol group and zwitterionic repeating groups; or a segmented multiblock polymer.

[0065] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer is at least partially crosslinked.

[0066] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protecting layer provides ionic strength, and the zwitterionic repeat compound is present in an amount capable of adjusting or maintaining the ionic strength. In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protecting layer provides free volume to allow reversible conformational change of one or more aptamer conjugates present sufficient to provide a detectable signal in the presence of an analyte.

[0067] In one embodiment, alone or in combination with any one of the preceding embodiments, an alkanethiol group or an arylthiol group is present at the end of a functionalized polymer chain. In one embodiment, alone or in combination with any one of the preceding embodiments, an alkanethiol group or an arylthiol group is present along the backbone of a functionalized polymer chain. In one embodiment, alone or in combination with any one of the preceding embodiments, an (aryl)mercaptoalkanol group is present at the end of a functionalized polymer chain. In one embodiment, alone or in combination with any one of the preceding embodiments, an (aryl)mercaptoalkanol group is present along the backbone of a functionalized polymer chain. In one embodiment, alone or in combination with any one of the preceding embodiments, a zwitterionic repeat group is present at the end of a functionalized polymer chain. In one embodiment, alone or in combination with any one of the preceding embodiments, a zwitterionic repeat group is present along the backbone of a functionalized polymer chain.

[0068] In one aspect, alone or in combination with any one of the preceding aspects, the one or more zwitterionic repeat groups comprise a betaine compound or a derivative thereof. In one aspect, alone or in combination with any one of the preceding aspects, the aptamer protection layer is configured to physically or chemically bind to at least a portion of the substrate surface.

[0069] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises a segmented multi-block polyurethane polymer.

[0070] In one aspect, alone or in combination with any one of the previous aspects, the segmented multi-block polymer comprises at least one of polyurethane, polyurea, poly(urethane urea), epoxide, polyolefin, polysiloxane, polyamide, polystyrene, polyacrylate, polyether, polyol, polyvinylpyridine, polyvinylpyrrolidone, polyester, polycarbonate, and copolymers thereof.

[0071] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises a segmented multi-block polyurethaneurea polymer.

[0072] In one embodiment, alone or in combination with any one of the previous embodiments, the segmented multiblock polymer comprises a soft segment and a hard segment. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment is hydrophobic or hydrophilic. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment is hydrophobic and hydrophilic. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment comprises a hydrophobic polyol and a hydrophilic polyol.

[0073] In one aspect, alone or in combination with any one of the preceding aspects, the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof. In one aspect, alone or in combination with any one of the preceding aspects, the soft segment is one or more segments comprising polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymer, and blends or copolymers thereof.

[0074] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer has an average molecular weight of about 1 kDa to about 500 kDa.

[0075] In another example, a method for determining the in vivo concentration of an analyte is provided, the method comprising contacting a biological fluid containing the analyte in vivo with an electrochemical aptamer biosensor bound to a conductive substrate, wherein an aptamer probe is encapsulated in an aptamer protection layer, the aptamer protection layer being permeable to the analyte, and wherein the electrochemical aptamer biosensor generates a signal upon interaction with the analyte; interrogating the conductive substrate or the electrochemical aptamer; and detecting a signal corresponding to the in vivo concentration of the analyte.

[0076] In one embodiment, alone or in combination with any one of the preceding embodiments, the interrogating is continuous, semi-continuous, sequential, or random detection of the signal. In one embodiment, alone or in combination with any one of the preceding embodiments, further includes adjusting the signal based on background signal generated as a result of non-specific binding of the aptamer biosensor to generate a regulated signal.

[0077] In one embodiment, alone or in combination with any one of the preceding embodiments, further comprising determining an in vivo concentration of the analyte over a period of time based on the adjusted signal. In one embodiment, alone or in combination with any one of the preceding embodiments, interrogating the conductive substrate comprises a different measurement technique.

[0078] In one aspect, alone or in combination with any one of the previous aspects, the differential measurement technique includes probing a conductive substrate at a first square wave voltammetry (SWV) frequency to obtain a first signal and at a second SWV frequency to obtain a second signal, taking the difference between the two signals, and dividing by the average of the two signals to obtain an adjusted signal.

[0079] In one embodiment, alone or in combination with any one of the preceding embodiments, the investigating comprises chronoamperometry. In one embodiment, alone or in combination with any one of the preceding embodiments, the investigating comprises cyclic voltammetry.

[0080] In one aspect, alone or in combination with any one of the previous aspects, the conductive substrate is an electrode, a microporous, or a nanoporous conductive material.

[0081] In another example, a method for manufacturing an electrochemical aptamer biosensor (EAB) is provided, the method comprising presenting at least one aptamer (at least one aptamer conjugate comprising a reversible redox moiety) on at least a portion of a surface of a conductive substrate, presenting an aptamer protection layer on the portion of the surface of the conductive substrate, and encapsulating at least a portion of the at least one aptamer conjugate in the aptamer protection layer.

[0082] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer further comprises one or more co-adsorbents. In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more co-adsorbents comprise a zwitterionic betaine group. In one embodiment, alone or in combination with any one of the preceding embodiments, the one or more zwitterionic betaine groups have the following structure:

[0083] [ka] (In the formula,

[0084] [ka] represents a hydrocarbon chain, and R1 and R2 are independently branched or unbranched acyclic alkyl, substituted or unsubstituted cyclic alkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocyclic, where X is -OH, -NHR1, -NH2, or -SH, and n is an integer from 2 to about 1000; or

[0085] [ka] wherein X is -OH, -NHR, -NH, or -SH; W, Y, and Z are independently branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, any of which may be optionally substituted with O, OH, halogen, amido, or alkoxyl; R is H, branched or unbranched acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; and R, R, and R are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl. In one embodiment, one or more of R1, R2, R3, R4, R5, and Z are covalently or ionically bonded to the APL.

[0086] In one embodiment, alone or in combination with any one of the preceding embodiments, the co-adsorbate is a terminally terminated dithiol having at least one betaine group along its chain. In one embodiment, alone or in combination with any one of the preceding embodiments, the thiol group of the terminally terminated dithiolalkanethiol is covalently bound to the substrate surface.

[0087] In one aspect, alone or in combination with any one of the previous aspects, the zwitterionic betaine group comprises a mercaptoalkanol betaine. In one aspect, alone or in combination with any one of the previous aspects, the mercaptoalkanol is linear and comprises multiple betaine groups along the chain.

[0088] In one embodiment, alone or in combination with any one of the preceding embodiments, the thiol group of the mercaptoalkanol is covalently attached to the substrate surface.

[0089] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises an alkanethiol group and one or more zwitterionic groups.

[0090] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer comprises a segmented multi-block polymer backbone. In one embodiment, alone or in combination with any one of the preceding embodiments, the segmented multi-block polymer backbone comprises a polyurethane polymer. In one embodiment, alone or in combination with any one of the preceding embodiments, the segmented multi-block polymer backbone comprises a polyurethane urea polymer.

[0091] In one embodiment, alone or in combination with any one of the previous embodiments, the segmented multiblock polymer comprises a soft segment and a hard segment. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment is hydrophobic or hydrophilic. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment is hydrophobic and hydrophilic. In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment comprises a hydrophobic polyol and a hydrophilic polyol.

[0092] In one aspect, alone or in combination with any one of the preceding aspects, the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof. In one aspect, alone or in combination with any one of the preceding aspects, the soft segment is one or more segments comprising polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, zwitterionic repeating group polymer, and blends or copolymers thereof.

[0093] In one embodiment, alone or in combination with any one of the preceding embodiments, the segmented multiblock polymer comprises a soft segment and a hard segment. In one embodiment, alone or in combination with any one of the preceding embodiments, the hard segment comprises a urethane group or a urea group.

[0094] In one embodiment, alone or in combination with any one of the previous embodiments, the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

[0095] In one embodiment, alone or in combination with any one of the preceding embodiments, the aptamer protection layer is crosslinked using an amount of a crosslinker.

[0096] In order to understand and see how the present disclosure may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0097] [Figure 1A] FIG. 1 is a schematic diagram showing an aptamer-protecting material used in a biosensor according to the broadest aspect of the present disclosure. [Figure 1B] FIG. 1 is a schematic diagram showing an aptamer-protecting material used in a biosensor according to the broadest aspect of the present disclosure. [Figure 2A] FIG. 1 is a schematic diagram illustrating an exemplary aptamer biosensor construct. [Figure 2B] 2B is a schematic diagram illustrating an alternative structure of the sensing region with an aptamer-protecting material of the exemplary aptamer biosensor shown in FIG. 2A. [Figure 2C] 2B is a schematic diagram illustrating an alternative structure of the sensing region with an aptamer-protecting material of the exemplary aptamer biosensor shown in FIG. 2A. [Figure 2D] 2B is a schematic diagram illustrating an alternative structure of the sensing region with an aptamer-protecting material of the exemplary aptamer biosensor shown in FIG. 2A. [Figure 2E] 1A-1C are a representative schematic diagram of a linear substrate aptamer construct with an aptamer-protecting material and a hypothetical graph of potential versus electrical double layer, respectively, in accordance with the present disclosure. [Figure 2F] 1A-1C are a representative schematic diagram of a linear substrate aptamer construct with an aptamer-protecting material and a hypothetical graph of potential versus electrical double layer, respectively, in accordance with the present disclosure. [Figure 2G] 1A-1C are a representative schematic of a microporous substrate aptamer construct with an aptamer-protecting material and a hypothetical graph of potential versus electrical double layer, respectively. [Figure 2H] 1A-1C are a representative schematic of a microporous substrate aptamer construct with an aptamer-protecting material and a hypothetical graph of potential versus electrical double layer, respectively. [Figure 3] FIG. 1 is a schematic diagram of an exemplary aptamer-protection material according to the broadest aspect of the present disclosure. [Figure 4A] FIG. 1 is a schematic diagram of an exemplary co-adsorbent according to the broadest aspect of the present disclosure. [Figure 4B] FIG. 1 is a schematic diagram of an exemplary co-adsorbent according to the broadest aspect of the present disclosure. [Figure 5A] 1A-1C are representative graphs of experimental charge versus frequency data for a control aptamer biosensor versus an exemplary aptamer biosensor with an aptamer-protecting material, respectively, in accordance with the present disclosure. [Figure 5B] 1A-1C are representative graphs of experimental charge versus frequency data for a control aptamer biosensor versus an exemplary aptamer biosensor with an aptamer-protecting material, respectively, in accordance with the present disclosure. [Figure 6A] 1A-1C are representative graphs of experimental charge versus frequency data of protein fouling for a control aptamer biosensor versus an exemplary aptamer biosensor with an aptamer protection material, respectively, in accordance with the present disclosure. [Figure 6B] 1A-1C are representative graphs of experimental charge versus frequency data of protein fouling for a control aptamer biosensor versus an exemplary aptamer biosensor with an aptamer protection material, respectively, in accordance with the present disclosure. [Figure 7A]1A-1C are representative graphs of experimental charge versus frequency data of protein fouling for a control aptamer biosensor versus an exemplary aptamer biosensor with an aptamer protection material, respectively, in accordance with the present disclosure. [Figure 7B] 1A-1C are representative graphs of experimental charge versus frequency data of protein fouling for a control aptamer biosensor versus an exemplary aptamer biosensor with an aptamer protection material, respectively, in accordance with the present disclosure. [Figure 8A] 1A-1C are representative graphs of experimental current versus frequency data for an exemplary aminoglycoside aptamer biosensor without and with, respectively, an aptamer-protecting material in accordance with the present disclosure. [Figure 8B] 1A-1C are representative graphs of experimental current versus frequency data for an exemplary aminoglycoside aptamer biosensor without and with, respectively, an aptamer-protecting material in accordance with the present disclosure. [Figure 9A] 1 is a representative graph of experimental normalized readout percentage versus time illustrating drift for a control aptamer biosensor versus exemplary aptamer biosensors with various aptamer protection materials exposed to proteins in accordance with the present disclosure. [Figure 9B] 1 is a representative graph of experimental normalized readout percentage versus time showing drift for an exemplary aptamer biosensor with an aptamer protection layer exposed to bovine serum albumin protein in accordance with the present disclosure. [Figure 9C] 1 is a representative graph of experimental normalized readout percentage versus time illustrating the stability of an exemplary aptamer biosensor having an aptamer protection layer according to the present disclosure. [Figure 10A] 1A-1C are representative graphs of experimental current versus frequency data for an exemplary vancomycin aptamer biosensor over time without and with, respectively, an aptamer-protecting material in accordance with the present disclosure. [Figure 10B]1A-1C are representative graphs of experimental current versus frequency data for an exemplary vancomycin aptamer biosensor over time without and with, respectively, an aptamer-protecting material in accordance with the present disclosure. [Figure 11] 1 is a representative graph of experimental sensor response percentage versus analyte concentration, depicting an exemplary aptamer biosensor with and without an aptamer-protection material exposed to various analyte concentrations, in accordance with the present disclosure. [Figure 12A] 1A-1C are representative graphs of exemplary vancomycin aptamer biosensors with different co-adsorbents, respectively, in accordance with the present disclosure. [Figure 12B] 1A-1C are representative graphs of exemplary vancomycin aptamer biosensors with different co-adsorbents, respectively, in accordance with the present disclosure. [Figure 13A] 1A-1C are representative graphs of shelf life performance of uncoated EABs versus exemplary APL-coated EABs, respectively, after 5 hours of ambient storage. [Figure 13B] 1A-1C are representative graphs of shelf life performance of uncoated EABs versus exemplary APL-coated EABs, respectively, after 5 hours of ambient storage. [Figure 13C] 1 is a representative graph of the calibration and drift performance of exemplary vancomycin APL coated EABs after one month of ambient storage. [Figure 13D] 1 is a representative graph of the calibration and drift performance of exemplary vancomycin APL coated EABs after one month of ambient storage. [Figure 13E] 1 is a representative graph of the calibration and drift performance of exemplary vancomycin APL coated EABs after two months of storage in an ambient dark environment. [Figure 13F] 1 is a representative graph of the calibration and drift performance of exemplary vancomycin APL coated EABs after two months of storage in an ambient dark environment. [Figure 14]FIG. 1 illustrates one particular embodiment of an exemplary continuous analyte monitoring sensor system in communication with at least one display device according to various techniques described in this disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0098] Despite significant progress toward implementing AB and EAB devices in vivo, significant challenges regarding aptamer stability must be overcome to facilitate continuous operation in complex samples such as blood or ISF. Novel EAB interfaces must be developed that can withstand continuous electrochemical investigations in biological fluids over extended periods of time—a process typically seen as a faradaic decrease and an increase in charging current over time. This degradation process limits the in vivo operational lifetime of EABs to 12 hours or less, a period much shorter than the elimination half-life of most drugs in humans. The present disclosure provides a technical solution to the above problem, facilitating continuous operation of AB and EAB devices in vivo using aptamer-protecting materials, either alone or in combination with co-adsorbents.

[0099] definition As used herein, the term "about" is a broad term and is to be given its ordinary and accustomed meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to allowing for a degree of variability in values ​​or ranges, for example, within 10%, within 5%, or within 1% of the stated limits of a stated value or range, including, but not limited to, the exactly stated value or range. As used herein, the term "substantially" refers to a majority or majority, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. As used herein, the phrase "substantially free" can mean having no or an insignificant amount of material such that the amount of material present does not affect the material properties of a composition that includes the material, such as about 0% to about 5% by weight of the composition being the material, or about 0% to about 1%, or about 5% by weight or less, or about 4.5% by weight or less, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001% by weight or less, or about 0% by weight.

[0100] As used herein, the terms "stick" and "adhere" are broad terms and are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, holding, joining, or fastening, for example, by adhering, bonding, grasping, interpenetrating, or fusing.

[0101] As used herein, the term "analyte" is a broad term and is given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning) and refers to, but is not limited to, a substance or chemical constituent in a biological fluid (e.g., blood, interstitial fluid, cerebrospinal fluid, lymphatic fluid, urine, sweat, saliva, etc.) that may be analyzed. Analytes may include naturally occurring substances, man-made substances, drugs, toxins, metabolites, and / or reaction products. Exemplary analytes include troponin, BNP, insulin, GLP-1, dopamine, serotonin, L-DOPA, vancomycin, aminoglycosides, doxorubicin, cortisol, and luteinizing hormone.

[0102] As used herein, the phrases “analyte measuring device,” “analyte monitoring device,” “analyte sensing device,” “continuous analyte sensing device,” “continuous analyte sensor device,” and / or “multi-analyte sensor device” are broad terms and are given their ordinary and customary meaning to those of skill in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, apparatus and / or systems responsible for detecting specific analytes or combinations of analytes or transducing signals associated therewith. For example, but not limited to, these terms may refer to instruments responsible for detecting specific analytes or combinations of analytes. In one example, the instrument includes a sensor coupled to a circuit arranged within a housing and configured to process signals associated with analyte concentrations into information. In one example, such devices and / or systems are capable of providing specific quantitative, semi-quantitative, qualitative, and / or semi-qualitative analytical information using biorecognition elements combined with transduction and / or detection elements.

[0103] As used herein, the term "aptamer" is a broad term and is given its ordinary and customary meaning to those skilled in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, an oligonucleotide or peptide that binds to a biological analyte. Aptamers can be of oligonucleotide or peptide origin. Oligonucleotide aptamers include nucleic acid species engineered through repeated rounds of in vitro selection or equivalently, SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind to biological analytes such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Peptide aptamers include polypeptides selected or engineered to bind to an analyte. Peptide aptamers can comprise or consist of one or more peptide loops of variable sequence displayed in a protein scaffold. Selection of peptide aptamers can be performed using different systems, including yeast two-hybrid systems, combinatorial peptide libraries constructed by phage display and other surface display technologies (e.g., mRNA display, ribosome display, bacterial display, and yeast display, collectively "biopanning"). Peptide aptamers can be selected from the MimoDB database. Peptide aptamers can also be isolated from combinatorial libraries created by directed mutation or variable region mutagenesis and selection rounds. Commercially available aptamers, including aptamers with transduction elements, can be purchased, for example, from Biosearch Technologies (Hoddesdon, UK).

[0104] As used herein, the phrase "aptamer conjugate" is a broad phrase and is given its ordinary and customary meaning to those skilled in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, an aptamer or bioactive agent covalently attached to a substrate, coadsorbate, carrier, or nanocarrier, such as a metal surface, a conductive surface, or a polymer, through a linker. The linker may be biologically inert so as to prevent the aptamer from dissociating from the substrate when exposed to or presented in a biological environment for a period suitable for continuous monitoring, such as in a wearable, or subcutaneous or transcutaneous environment with or without a protective layer. The linker may be biologically active so as to allow dissociation of the bioactive agent (e.g., an anti-inflammatory agent) from the carrier when exposed to or presented in a biological environment, such as a subcutaneous or transcutaneous environment. The phrase "aptamer conjugate" includes aptamers that include a linking moiety for binding or tethering to a substrate, coadsorbate, carrier, or nanocarrier, and also includes aptamers that include a linking moiety and a redox moiety attached thereto.

[0105] As used herein, the terms "aptamer protective material," "aptamer protective domain," "aptamer protective membrane," "aptamer protective region," "aptamer protective matrix," and "aptamer protective layer," collectively referred to as "aptamer protective layer 105" or "APL," are broad terms that are to be given their ordinary and customary meaning to those of skill in the art (and are not limited to any special or customized meaning) and refer to, but are not limited to, any substance, domain, membrane, region, polymer, matrix, or layer that functions in conjunction with one or more aptamer conjugates configured to transduce a signal corresponding to the concentration of a biological analyte. For example, the APL provides one or more of the following attributes: allows the aptamer conjugate to undergo conformational transformation within the APL; allows transport of one or more analytes; provides an electrochemical and / or physicochemical environment around the aptamer to stabilize the aptamer itself, its binding to the substrate, or the lifetime of a redox moiety attached to the aptamer; and reduces or eliminates signal drift in vivo over time.

[0106] As used herein, the phrases and terms "bioactive agent" and "bioactive" are broad phrases and terms that are to be given their ordinary and customary meaning to those of skill in the art (and are not limited to any special or customized meaning) and refer to any substance that has an effect on or elicits a response from living tissue, such as, but not limited to, drugs, biologics, reactive oxygen scavengers (ROS), and metal ions.

[0107] As used interchangeably herein, the phrases "biointerface membrane," "biointerface domain," and "biointerface layer" are broad terms that are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, a permeable membrane (which may include multiple domains) or layer that serves as a bioprotective interface between host tissue and an implantable device. The terms "biointerface" and "bioprotection" are used interchangeably herein.

[0108] As used herein, the terms "biosensor" and / or "sensor" are broad terms and are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, a portion of an analyte measuring device, analyte monitoring device, analyte sensing device, continuous analyte sensing device, sequential analyte sensing device, and / or multi-analyte sensing device responsible for detecting a specific analyte or combination of analytes or transducing a signal associated therewith. In embodiments, a biosensor or sensor generally comprises a body and working, reference, and / or counter electrodes coupled to the body and forming a surface configured to provide a signal during an electrochemical reaction. One or more membranes may be affixed to the body and cover the electrochemically reactive surface. In embodiments, such biosensors and / or sensors are capable of providing a specific quantitative, semi-quantitative, qualitative, or semi-qualitative analytical signal using a biorecognition element combined with a detection and / or transduction element.

[0109] As used herein, the term "biostable" is a broad term that is to be given its ordinary and customary meaning to those of skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, materials that are relatively resistant to degradation by processes encountered in vivo.

[0110] As used herein, the term "coadsorbate" is a broad term and is to be given its ordinary and customary meaning to those of skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, a material that adsorbs to, associates with, or binds to a substrate surface (adsorbent) through covalent, ionic, or molecular interactions. Unless otherwise specified, a coadsorbate is at least partially adsorbed onto a surface rather than absorbed onto the surface.

[0111] As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0112] As used herein, the term "continuous" is a broad term and is to be given its ordinary and customary meaning to those of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, an uninterrupted or unbroken portion, domain, coating, or layer.

[0113] As used herein, the phrase "continuous analyte sensing" is a broad phrase that is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning) and refers to, but is not limited to, continuous, continuous, and / or intermittent (but periodic) monitoring of analyte concentration, for example, for periods of time that are performed from about 5 seconds or less to about 10 minutes or more. In further embodiments, continuous monitoring of the analyte concentration is performed every about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds to about 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, 2.75, 3.00, 3.25, 3.50, 3.75, 4.00, 4.25, 4.50, 4.75, 5.00, 5.25, 5.50, 5.75, 6.00, 6.25, 6.50, 6.75, 7.00, 7.25, 7.50, 7.75, 8.00, 8.25, 8.50, 8.75, 9.00, 9.25, 9.50, or 9.75 minutes. In a further example, continuous monitoring of analyte concentration can be performed daily and can be performed over a period of several weeks.

[0114] As used herein, the term "coupled" is a broad term and is to be given its ordinary and customary meaning to those of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, two or more system elements or components that are configured to be electrically, mechanically, thermally, operatively, chemically, or otherwise attached to at least one other. Similarly, as used herein, the phrases "operably connected," "operably linked," and "operably coupled" can refer to one or more components that are coupled to another component in a manner that facilitates the transmission of at least one signal between the components. In some examples, the components are part of the same structure and / or are integrated with one another (i.e., "directly coupled"). In other examples, the components are connected via remote means. For example, one or more electrodes can be used to detect analytes in a sample and convert that information into a signal, which can then be transmitted to an electronic circuit. In this example, the electrodes are "operably coupled" to the electronic circuit. As used herein, the phrase "removably coupled" may refer to two or more system elements or components that are configured or configured to be electrically, mechanically, thermally, operatively, chemically, or otherwise attached and detached without damaging any of the coupled elements or components. As used herein, the phrase "permanently coupled" may refer to two or more system elements or components that are configured or attached electrically, mechanically, thermally, operatively, chemically, or otherwise attached, but cannot be separated without damaging at least one of the coupled elements or components.

[0115] As used herein, the term "discontinuous" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, cut, interrupted, or separated portions, layers, coatings, or domains.

[0116] As used herein, the term "distal" is a broad term and is to be given its ordinary and customary meaning to those skilled in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, an area that is relatively far away from a reference point such as an origin or attachment point.

[0117] As used herein, the term "domain" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, a region of a membrane system that may be a layer, a uniform or non-uniform gradient (e.g., an anisotropic region of a membrane), or a portion of a membrane that is capable of sensing one, two, or more analytes. Domains discussed herein can be formed as a single layer, as two or more layers, as a pair of bilayers, or as combinations thereof.

[0118] As used herein, the term "drift" is a broad term and is to be given its ordinary and customary meaning to those skilled in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, a gradual increase or decrease in signal over time that is unrelated to changes in host systemic analyte concentration. Without wishing to be bound by theory, it is believed that drift may be the result of a local decrease in analyte transport to the sensor, for example, due to the formation of a foreign body capsule (FBC). It is also believed that an insufficient amount of interstitial fluid surrounding the sensor may reduce transport to the sensor. In one example, an increase in local interstitial fluid may slow or reduce drift, thus improving sensor performance. Drift may also be the result of sensor electronics or algorithmic models used to compensate for noise or other anomalies that may occur with electrical signals in ranges including the milliampere, microampere, picoampere, nanoampere, and femtoampere ranges, as well as faradic, capacitance, and potential measurements.

[0119] The phrases "bioactive release film," "drug-release layer," "bioactive release domain," and "bioactive agent-releasing film" are used interchangeably herein and are each broad terms, given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, a permeable or semi-permeable film that is permeable to one or more bioactive agents. In embodiments, the "bioactive release film," "drug-release layer," and "bioactive release domain" and "bioactive agent-releasing film" can comprise two or more domains and are typically several microns or more thick. In embodiments, the bioactive release film and / or bioactive agent-releasing film and / or bioactive agent-releasing film are substantially the same as the biointerface layer and / or biointerface membrane. In other embodiments, the bioactive release film and / or bioactive release film and / or bioactive agent-releasing film and / or bioactive agent-releasing film are different from the biointerface layer and / or biointerface membrane.

[0120] As used herein, the term "electrochemically reactive surface" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, the surface of an electrode on which an electrochemical reaction occurs. In another example, electron transfer is provided using a redox moiety associated with an aptamer conjugate, where the redox moiety is capable of undergoing reduction-oxidation (redox) associated with a reversible binding interaction between the aptamer and the analyte that is proportional to the analyte concentration.

[0121] As used herein, the term "gain" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, the differential measurement between a signal OFF state and a signal ON state. For example, a typical gain range is 1-200% of the percentage change in signal caused by a particular concentration of analyte compared to zero analyte concentration. Analyte concentrations are typically quantified in micromoles (uM), nanomoles (nM), nanograms per milliliter (ng / mL), or picograms per milliliter (pg / mL).

[0122] As used herein, the phrase "hard segment" is a broad phrase and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, an element of a copolymer, such as a polyurethane, polycarbonate polyurethane, or polyurethane urea copolymer, that imparts resistance properties, such as resistance to bending or twisting. The term "hard segment" can be further characterized as a crystalline, semi-crystalline, or glassy material that has a glass transition temperature, typically determined by dynamic scanning calorimetry ("Tg"), above ambient temperature, and is typically made from a diisocyanate, with or without a chain extender.

[0123] As used herein, the term "host" is a broad term and is given its ordinary and customary meaning to those of skill in the art (and is not limited to any special or customized meaning), and refers to mammals, such as, but not limited to, humans.

[0124] As used herein, the terms "implanted" or "implantable" are broad terms that are to be given their ordinary and customary meaning to those skilled in the art (and are not to be limited to any special or customized meaning) and refer to an object (e.g., a sensor) that is inserted subcutaneously (i.e., within the fatty layer between the skin and the muscle) or percutaneously (i.e., penetrating, entering, or passing through intact skin), which may result in a sensor having an in vivo portion and an ex vivo portion, but is not limited to such.

[0125] As used herein, the terms "interferent" and "interfering species" are broad terms and are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, effects and / or species that interfere with the measurement of an analyte of interest in a sensor, producing a signal that does not accurately represent the analyte measurement. In the example of an electrochemical aptamer sensor, an interfering species is a compound that has a redox (reduction-oxidation) potential that overlaps with one or more redox moieties associated with the analyte being measured or one or more aptamers.

[0126] As used herein, the term "in vivo" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), encompassing, but not limited to, portions of a device (e.g., a sensor) adapted for insertion into and / or presence within the body of a host.

[0127] As used herein, the term "ex vivo" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), encompassing, but not limited to, a portion of a device (e.g., a sensor) that is adapted to reside and / or exist outside the host organism.

[0128] The term "linker," as used herein, is a broad term and is to be given its ordinary and customary meaning to one of skill in the art (and is not limited to any special or customized meaning), including, but not limited to, a chemical group or molecule that links two molecules or moieties, such as an aptamer and a substrate, coadsorbate, carrier, or nanocarrier. In one example, the linker is positioned between or adjacent to two groups, molecules, or other moieties and is connected to each other via a covalent bond, thus connecting the two. In one example, the linker is an oligonucleotide, biotin, maleimide (NHS) ester, polyethylene glycol-NHS ester, or "click" chemistry building block. In one example, thymidine nucleotides of 2 to 10 in length, with or without a spacer group, are used.

[0129] As used herein, the term "membrane" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to a structure configured to perform functions including, but not limited to, protecting exposed electrode surfaces from the biological environment, resisting (limiting) the diffusion of analytes, acting as a matrix for catalysts to enable enzymatic reactions, limiting or screening interfering species, providing hydrophilicity at electrochemically reactive surfaces of a sensor interface, acting as an interface between host tissue and an implantable device, modulating host tissue response through drug (or other substance) release, and combinations thereof. As used herein, the terms "membrane" and "matrix" are meant to be interchangeable.

[0130] As used herein, the phrase "membrane system" is a broad phrase and is to be given its ordinary and customary meaning to one of skill in the art (and is not limited to any special or customized meaning), and refers, without limitation, to a permeable or semi-permeable membrane that may be composed of two or more domains, layers, or layers within domains, is typically composed of material several microns or more in thickness, and is permeable to an analyte. In embodiments, the membrane system includes an immobilized or encapsulated aptamer that allows a conversion to occur between the aptamer and the analyte, thereby enabling the analyte concentration to be measured.

[0131] As used herein, the term "micro" is a broad term and is to be given its ordinary and customary meaning to those skilled in the art (and is not to be limited to any special or customized meaning), and refers to a size of approximately 10 microns that is not visible without magnification. -6 "Micro" refers to objects or scales that are small, but not limited to, those on the order of 10 microns. The term "micro" is in contrast to the term "macro," which refers to objects that are large enough to be seen without magnification. Similarly, the term "nano" refers to objects that are approximately 10 -9 Refers to a small object or scale of m.

[0132] As used herein, the term "noise" is a broad term and is used in its ordinary sense to include, but is not limited to, signals detected by a sensor or sensor electronics that are unrelated to analyte concentration and may result in degraded sensor performance. Some types of noise are observed for several hours (e.g., about 2 to about 24 hours) after sensor insertion. After the first 24 hours, noise may disappear or diminish, but in some hosts, noise may persist for about 3 to 4 days. In some cases, noise can be reduced using predictive modeling, artificial intelligence, and / or algorithmic means. In other cases, noise can be reduced by addressing immune response factors associated with the presence of an implanted sensor, such as by using a bioactive-releasing membrane with at least one bioactive agent. For example, the noise of one or more exemplary biosensors as disclosed herein can be determined and then compared qualitatively or quantitatively. As an example, by acquiring a raw signal time series at a fixed sampling interval (in picoamperes (pA)), a smoothed version of the raw signal time series can be obtained, for example, by applying a third-order low-pass digital Chebyshev Type II filter. Other smoothing algorithms can also be used. At each sampling interval, the absolute difference in pA can be calculated to provide a smoothed time series. This smoothed time series can be converted to units (units of "noise") using, for example, an analyte sensitivity time series derived by using a mathematical model between the raw signal and reference blood analyte measurements. Optionally, the time series can be aggregated, for example, by hour or day, as desired. Comparison of corresponding time series between different exemplary biosensors having a bioactive-releasing membrane and one or more bioactive agents of the present disclosure provides a qualitative or quantitative determination of noise improvement.

[0133] As used herein, the terms "optional" or "optionally" are broad terms and are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any special or customized meaning), meaning, without limitation, that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not occur.

[0134] As used herein, the phrase "polymerizable group" is a broad phrase and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, a functional group that allows for the polymerization of a monomer with itself to form a homopolymer, or with a different monomer to form a copolymer. Depending on the type of polymerization method used, the polymerizable group may be selected from alkenes, alkynes, epoxides, lactones, amines, hydroxyls, isocyanates, carboxylic acids, anhydrides, silanes, halides, aldehydes, and carbodiimides.

[0135] As used herein, the term "polyzwitterion" is a broad term and is given its ordinary and customary meaning to those skilled in the art (and is not limited to any special or customized meaning), referring to, but not limited to, polymers in which the repeat units of the polymer chain are zwitterionic moieties. Polyzwitterions are also known as polybetaines. Because polyzwitterions have both cationic and anionic groups, they are a type of polyampholytic polymer. However, they are unique because both the cationic and anionic groups are part of the same repeat unit, meaning that polyzwitterions have the same number of cationic and anionic groups, whereas other polyampholytic polymers can have more of one ionic group than the other. Polyzwitterions also have cationic and anionic groups as part of the repeat unit. A polyampholytic polymer need not have cationic groups attached to anionic groups; they can be on different repeat units and thus distributed apart from each other at random intervals, or one ionic group can outnumber the other.

[0136] As used herein, the term "proximal" is a broad term and is given its ordinary and customary meaning to those skilled in the art (and is not limited to any special or customized meaning), referring to, but not limited to, the spatial relationship between various elements compared to a particular reference point. For example, some examples of devices include a membrane system having a biological interface layer and an enzyme layer. If the sensor is considered to be the reference point and the enzyme layer is positioned closer to the sensor than the biological interface layer, then the enzyme layer is more proximal to the sensor than the biological interface layer.

[0137] As used herein, the phrases and terms "processor module" and "microprocessor" are each broad phrases and terms that are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning) and refer to, but are not limited to, a computer system, state machine, processor, or the like, designed to perform arithmetic or logical operations using logic circuitry that responds to and processes the basic instructions that drive a computer.

[0138] As used herein, the term "semi-continuous" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, a portion, coating, domain, or layer that includes one or more continuous and discontinuous portions, coatings, domains, or layers. For example, a coating that is disposed around but not over a sensing area is "semi-continuous."

[0139] As used herein, the phrases "sensing moiety," "sensing membrane," "sensing region," "sensing domain," and / or "sensing mechanism" are broad terms and are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, a portion of a biosensor and / or sensor responsible for detecting or transducing a signal associated with a particular analyte or combination of analytes. In examples, the sensing moiety, sensing membrane, and / or sensing mechanism generally comprise electrodes configured to provide a signal during an electrochemical reaction with one or more membranes covering an electrochemically reactive surface. In examples, such sensing moieties, sensing membranes, and / or sensing mechanisms are capable of providing a specific quantitative, semi-quantitative, qualitative, or semi-qualitative analytical signal using a biorecognition element in combination with a detection and / or transduction element.

[0140] During typical operation of an analyte measuring device, biosensor, sensor, sensing region, sensing moiety, or sensing mechanism, a biological sample, e.g., blood or interstitial fluid, or a component thereof, either directly or after passing through one or more membranes, contacts an aptamer, or an RNA or DNA protein, or, for example, one or more periplasmic binding proteins (PBPs) or variants or fusion proteins thereof, having one or more analyte-binding regions, each region capable of specifically and reversibly binding at least one analyte. The interaction of the biological sample or a component thereof with the analyte measuring device, biosensor, sensor, sensing region, sensing moiety, or sensing mechanism results in the transduction of a signal that allows for a qualitative, semi-qualitative, quantitative, or semi-qualitative determination of the analyte level in the biological sample.

[0141] In examples, the sensing region or sensing portion may comprise at least a portion of a conductive substrate, or at least a portion of a conductive surface, e.g., a substantially planar substrate including wires or conductive traces, or substantially planar traces, and a membrane. In examples, the sensing region or sensing portion may comprise a non-conductive body, working, reference, and counter electrodes (optional) that form an electrochemically reactive surface at one location on the body and form electronic connections at another location on the body, and a sensing membrane affixed to the body and covering the electrochemically reactive surface.

[0142] In one example, multiple working electrodes can be used. For example, a second working electrode comprising aptamer conjugates for multiple different analytes (e.g., analyte 1, analyte 2, etc.) on the second working electrode to compensate for sensor drift and / or interference. Similarly, a second working electrode comprising non-selective aptamer conjugates for multiple different analytes (e.g., analyte 1, analyte 2, etc.) on the second working electrode can be used to compensate for sensor drift and / or interference.

[0143] In one example, a combination of at least two sets of identical aptamers, but one set with a different redox moiety, is used to correct for sensor drift and / or interference. In one example, a combination of at least two sets of non-identical aptamer conjugates (e.g., different linkers / linker lengths, binding chemistries, different selectivities, and / or binding affinities), each set with the same redox moiety, is used to correct for sensor drift and / or interference and / or provide detection within a wide physiological analyte concentration range. In one example, a combination of at least two sets of non-identical aptamer conjugates (e.g., different linkers / linker lengths, binding chemistries, different selectivities, and / or binding affinities), each set with a unique redox moiety, is used to correct for sensor drift and / or interference and / or provide detection within a wide physiological analyte concentration range. In one example, the same or different aptamers are conjugated to different redox moieties with separated formal potentials to reduce or eliminate signal from interfering species.

[0144] In another example, the sensing region can include one or more periplasmic binding proteins (PBPs) or their variants or fusion proteins having one or more analyte-binding regions, each capable of specifically and reversibly binding at least one analyte. Mutations in the PBPs can contribute to or alter one or more of the following: binding constant, extended stability of the protein, including thermal stability, to bind the protein to a specific encapsulation matrix, membrane, or polymer, or to attach a detectable reporter group or "label" to indicate changes in the binding region. Specific examples of changes in the binding region include, but are not limited to, changes in the hydrophobic / hydrophilic environment, three-dimensional conformational changes, changes in the orientation of amino acid side chains in the protein's binding region, and the redox state of the binding region. Such changes to the binding region provide a detectable signal transduction corresponding to one or more analytes present in the biological fluid.

[0145] In embodiments, the sensing region determines selectivity between one or more analytes such that only the analyte that must be measured results in (transduces) a detectable signal. This selection can be based on any chemical or physical recognition of the analyte by the sensing region, where the chemical composition of the analyte is not changed, or where the sensing region causes or catalyzes a reaction of the analyte that changes the chemical composition of the analyte.

[0146] The sensing region converts the recognition of the analyte into a semi-quantitative or quantitative signal. Thus, as used herein, "transducing" or "transduction" and their grammatical equivalents encompass optical, electrochemical, acoustic / mechanical, or colorimetric techniques and methods. Electrochemical properties include current and / or voltage, capacitance, and electric potential. Optical properties include absorbance, fluorescence / phosphorescence, wavelength shift, phase modulation, bio / chemiluminescence, reflectance, light scattering, and refractive index.

[0147] As used herein, the term "sensitivity" is a broad term and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers, without limitation, to the amount of signal (e.g., in the form of current and / or voltage) produced by a given amount (unit) of analyte measured. For example, an amperometric sensor has a sensitivity (or slope) of about 1 to about 100 picoamps of current per 1 mg / dL of analyte.

[0148] As used herein, the phrases and terms "small diameter sensor," "miniature structured sensor," and "microsensor" are broad phrases and terms that are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, a sensing mechanism having at least one dimension that is less than about 2 mm. In further embodiments, the sensing mechanism has at least one dimension that is less than about 1 mm. In some embodiments, the sensing mechanism (sensor) is less than about 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mm. In some embodiments, the largest independently measured dimension of length, width, diameter, thickness, or circumference of the sensing mechanism does not exceed about 2 mm. In some embodiments, the sensing mechanism is a needle-type sensor having a diameter of less than about 1 mm; see, e.g., U.S. Pat. No. 6,613,379 to Ward et al. and U.S. Pat. No. 7,497,827 to Brister et al., both of which are incorporated by reference in their entireties. In some alternative embodiments, the sensing mechanism includes an electrode deposited on a substantially planar substrate, and the thickness of the implantable portion is less than about 1 mm; see, e.g., U.S. Pat. No. 6,175,752 to Say et al. and U.S. Pat. No. 5,779,665 to Mastrototaro et al., both of which are incorporated by reference in their entireties. Examples of sensors (sensor electrode layouts and films) and methods of forming sensor systems discussed herein can be found in currently pending U.S. Patent Application Publication No. 2019-0307371, which is incorporated by reference in its entirety.

[0149] As used herein, the phrase "soft segment" is a broad phrase and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not limited to any special or customized meaning), and refers to, but is not limited to, an element of a copolymer, such as, for example, a polyurethane, a polycarbonate polyurethane, or a polyurethane urea copolymer, that imparts flexibility to the chain. The phrase "soft segment" can be further characterized as an amorphous material having a low Tg, e.g., a Tg that is typically no higher than ambient temperature or normal mammalian body temperature.

[0150] As used herein, the phrase "solid portion" is a broad phrase that is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers to, but is not limited to, the portion of the material of the membrane that has a mechanical structure that defines a cavity, void, or other non-solid portion.

[0151] As used herein, the terms "zwitterionic" and "zwitterionic compound" are broad terms and phrases, respectively, that are to be given their ordinary and customary meaning to one of ordinary skill in the art (and are not to be limited to any special or customized meaning), and refer to, but are not limited to, compounds in which a neutral molecule of the compound has a unit positive charge and a unit negative charge at different locations within the molecule. Such compounds are a type of zwitterionic compound and are sometimes referred to as "inner salts."

[0152] As used herein, the phrases "zwitterionic precursor" or "zwitterionic compound precursor" are broad terms and are to be given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, any compound that is not itself zwitterionic but can become zwitterionic in a final or transition state through a chemical reaction. In some examples described herein, the devices include zwitterionic precursors that can be converted to zwitterionic form prior to in vivo implantation of the device. Alternatively, in some examples described herein, the devices include zwitterionic precursors that can be converted to zwitterionic form through several chemical reactions that occur after in vivo implantation of the device. Such reactions are known to those skilled in the art and include ring-opening reactions, addition reactions such as Michael addition, and the like. This method is particularly useful when polymerization of betaine-containing monomers is difficult due to technical challenges, such as the solubility of the betaine monomer to achieve desired physical properties, such as molecular weight and mechanical strength. Post-polymerization modification or conversion of betaine precursors can be a practical method for achieving desired polymer structures and compositions. Examples of such precursors include tertiary amines, quaternary amines, pyridine, and others detailed herein.

[0153] As used herein, the phrases "zwitterionic derivative" or "zwitterionic compound derivative" are broad terms and are given their ordinary and customary meaning to those skilled in the art (and are not limited to any special or customized meaning), and refer to, but are not limited to, any compound that is not itself zwitterionic, but rather is the product of a chemical reaction in which a zwitterionic compound is converted to a non-zwitterionic compound. Such a reaction can be reversible, such that under certain conditions, the zwitterionic derivative can act as a zwitterionic precursor. For example, a hydrolyzable betaine ester formed from a zwitterionic betaine is a cationic zwitterionic derivative that can undergo hydrolysis back to the zwitterionic betaine under appropriate conditions.

[0154] As used herein, the phrase "zwitterionic repeat group" is a broad phrase and is to be given its ordinary and customary meaning to one of ordinary skill in the art (and is not to be limited to any special or customized meaning), and refers, without limitation, to two or more zwitterionic compounds, zwitterionic derivatives, or zwitterionic compound derivatives, independently, in the same compound or polymer.

[0155] Aptamer-based biosensors (ABs) and electrochemical aptamer-based biosensors (EABs) are analytical platforms that can provide continuous in vivo monitoring of specific molecular analytes. EAB sensors typically exhibit an architecture consisting of a self-assembled monolayer (SAM) of analyte-binding alkanethiol-functionalized nucleic acid aptamers or other bioreceptors containing a sensitive redox moiety as a signal transduction element to correlate analyte binding events with measurable electrical energy changes, and an SAM of an alkanethiol-based electrode-blocking co-adsorbent to prevent unwanted electrochemical reactions and confer biocompatibility to the electrode surface.

[0156] The poor stability observed when the above-described ABs or EABs are placed in physiologically relevant environments is due, at least in part, to detachment of the aptamer monolayer from the substrate surface or the underlying SAM monolayer used to immobilize the aptamer or electrode-blocking SAM, as well as degradation (e.g., fouling, drift, etc.) of the bioelectronic interface during continuous electrochemical probing, a process typically seen as a faradaic decrease and an increase in charging current over time. As discussed in more detail below, such performance deficiencies can be addressed with the aptamer protection layer (APL) of the present disclosure.

[0157] In one embodiment, the present disclosure provides an AB or EAB in an architecture consisting of a self-assembled monolayer (SAM) of an analyte-binding alkanethiol or carboxyl-functionalized nucleic acid aptamer or other bioreceptor, comprising a signal transduction element for correlating the analyte binding event with a measurable signal from the transduction element, a SAM of an alkanethiol and / or functionalized alkanethiol electrode-blocking co-adsorbent, and an aptamer protective layer (APL) adjacent or directly adjacent to the architecture, which, independently or collectively, prevents unwanted desorption, unwanted reactions, reduces biofouling / confers biocompatibility, aptamer stability, and device longevity.

[0158] In one example, the aptamer conjugate and APL are temperature controlled during use, e.g., the wearable sensor is insulated and / or configured with a mini-Peltier cooler and / or a heat exchange device, e.g., fins, or a combination of the above. In another example, the aptamer is prepared under conditions that closely match the in vivo thermodynamic environment of the sensor (e.g., Systematic Evolution of Ligands by Exponential Enrichment (SELEX)), thereby providing or improving high affinity and / or thermal stability.

[0159] 1A and 1B, an exemplary aptamer-based analyte monitoring sensor 100 configured for in vivo measurement of at least one analyte 99 is presented along with a schematic diagram showing an aptamer-protecting material 105. An aptamer 102 having a signal transduction element 104 is shown associated with an optional monolayer 103 adjacent to a substrate 110. The monolayer 103 can be covalently or non-covalently bound to the substrate 110. In one example, the aptamer 102 undergoes a reversible conformational change upon interaction with the analyte 99, e.g., an analyte, a metabolite, a drug, etc., causing the signal transduction element 104 to be presented closer to the substrate 110 so as to provide a signal corresponding to the concentration or presence of the analyte 99.

[0160] In one example, the signal transduction element 104 is a reversible redox moiety, the substrate 110 is conductive, and the reversible binding (and subsequent reversible conformational change) of the aptamer 102 upon interaction with the analyte 99 causes a change in proximity of all or a portion of the signal transduction element 104 to that of the conductive substrate 110, such that the signal transduction element 104 can undergo a detectable reversible reduction-oxidation reaction via electron transfer with the conductive substrate 110 upon reversible binding of the analyte 99 with the aptamer 102. The detectable reversible reduction-oxidation reaction via electron transfer with the conductive substrate 110 provides a correlation with the concentration of the analyte 99, as discussed further below.

[0161] In another example, reversible binding (and subsequent reversible conformational change) of the aptamer 102 upon interaction with the analyte 99 can cause all or a portion of the signal conversion element 104 to be presented in or relative to a different local environment, for example, from a hydrophobic local environment to a hydrophilic local environment (or vice versa), so as to provide a detectable signal corresponding to the concentration or presence of the analyte 99.

[0162] In one example, the signal transduction element 104 is an environmentally sensitive fluorescent or phosphorescent dye that can undergo a detectable change in emission wavelength or frequency and / or emission relaxation or decay rate upon exposure to electromagnetic radiation, e.g., light, e.g., upon reversible binding with the analyte 99 and a reversible conformational change from a hydrophobic local environment to a hydrophilic local environment (or vice versa), the detectable change in emission wavelength or frequency and / or emission relaxation or decay rate to provide a correlation with the concentration of the analyte 99.

[0163] The signal transduction element 104 can be covalently or non-covalently bound to the aptamer 102, where the covalent or non-covalent binding is sufficient for continuous signal transduction of a signal over a period of time commensurate with a transdermal, intradermal, subcutaneous, ocular, or skin-based continuous analyte sensing device. In one example, continuous signal transduction of a signal over a period of at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 1 week, at least 2 weeks, or at least 3 weeks is contemplated using the aptamer-protecting material 105 of the present disclosure.

[0164] In one example, the signal transduction element 104 and the aptamer 102 are conjugated or form a conjugate. In one example, the conjugate of the signal transduction element 104 and the aptamer 102 is associated with the monolayer 103. In one example, the conjugate of the signal transduction element 104 and the aptamer 102 is covalently or non-covalently bound to the monolayer 103. In one example, the conjugate of the signal transduction element 104 and the aptamer 102 is covalently or non-covalently bound to the substrate 110.

[0165] For example, when referring hereinafter to a redox moiety as the signal transduction element 104, this is for the sake of brevity and does not limit the scope of the signal transduction element 104. Thus, "redox moiety 104" and "signal transduction element 104" are used interchangeably hereinafter.

[0166] FIG. 2A is a schematic diagram illustrating an exemplary aptamer biosensor 200 construct configured for continuous in vivo use in a subject. Accordingly, biosensor 200 is shown as an elongated member having a sensing region 207 fabricated, for example, from a window in an electrically insulating coating 205 around a conductive wire. Alternatively, a window may be prepared within the jacket of an optical fiber for use with an optically-based AB device. Additional electrodes 215 (reference and / or counter electrodes) may be used separately or provided, for example, as adjacent coaxial elongated members. As shown in enlarged cross-sectional views 2B-2D, alternative structures 201, 202, and 203 of sensing region 207 are shown in conjunction with a substrate 110 surface; for example, structure 201 has a substrate 110 surface with adjacent aptamers 102 and aptamer-protecting material 105. Structure 202 has a substrate 110 surface with adjacent co-adsorbates 103, aptamers 102, and aptamer protection material 105. Structure 203 has a substrate 110 surface adjacent to the co-adsorbates 103, aptamers 102, aptamer protection material 105, and a drug-releasing film 113 most distal from the substrate 110. Other configurations of co-adsorbates 103, aptamers 102, aptamer protection material 105, and drug-releasing film 113 can be used.

[0167] Substrate / Electrode In an embodiment, the substrate 110 surface corresponds to an AB or EAB pattern for use in a continuous sensing device. In one embodiment, the substrate 110 is or includes a conductive material. In one embodiment, the substrate 110 is an electrode, which may be a wire, a planar structure, or a substantially planar structure. In one embodiment, the substrate 110 can be configured to independently provide one or more of a working electrode, a reference electrode, and optionally a counter electrode. In one embodiment, one or more of the working electrode, the reference electrode, and optionally a counter electrode are arranged in a linear or substantially linear configuration. In one embodiment, the reference electrode includes silver (Ag) and / or silver chloride (AgCl). In one embodiment, the reference electrode includes silver (Ag) and / or silver chloride (AgCl) encapsulated or otherwise coated with a protective layer. In one embodiment, the reference electrode with a protective layer prevents AgCl from escaping from the reference electrode.+ , Ag, AgCl -2 The protective layer is configured to reduce or eliminate diffusion of ions or particles and / or reduce or eliminate interaction of the reference electrode or AgCl ions with the aptamer and / or thiol-containing coadsorbate or thiol-containing aptamer protective layer. In one example, the protective layer of a silver reference electrode is configured to inhibit or reduce transport of AgCl ions while allowing transport of chloride ions. Examples of suitable protective layers for silver reference electrodes include, but are not limited to, amphiphilic polyurethane or polyurethane urea, Teflon, microporous Teflon, ion-selective membranes, semipermeable membranes, PVC, and plasticized PVC.

[0168] In one embodiment, the substrate 110 comprises a wire formed from or coated with a conductive material such as platinum, platinum-iridium, palladium, graphite, gold, carbon, graphene, graphene oxide, a conductive polymer, or an alloy.

[0169] In one embodiment, at least a portion of the substrate 110 comprises pores having an average pore size of nanometer and / or micrometer dimensions. Such pore sizes in the substrate can be formed, for example, using etching or plasma techniques. Substrates having such nanometer and / or micrometer dimensions can be used in combination with the APLs of the present disclosure.

[0170] For example, the structural properties of the substrate can be limiting in EAB performance. As shown in Figures 2E and 2F, a planar substrate 110 bearing aptamers 102 and attached redox moieties 104 exhibits the potential versus electric double layer (EDL) relationship shown, the area contained within the Debye volume. In contrast, Figure 2G shows the same substrate 222 with an aptamer 102 and attached redox moieties 104 construct with at least some nanometer- and / or micrometer-sized pores 225 at its surface. As shown in Figure 2H, the potential versus electric double layer (EDL) relationship exhibits a smaller relative negative slope compared to a linear substrate. When used in combination with the APLs of the present disclosure, the substrate 222 surface can provide increased signal and detection limits for continuous EAB devices. Additionally, the above constructs can provide for intercalation of redox moieties between closely spaced aptamers, which can provide two sites for absorption of two analytes via (pi-pi)π-π interactions of selected analytes-redox moieties (e.g., methylene blue and dopamine) rather than absorption of one analyte by the aptamer alone, thereby doubling the detection sensitivity.

[0171] In one example, the substrate 110 surface comprises a conductive surface, and at least a portion of the substrate 110 surface is configured to covalently bond, tether, or associate with an aptamer conjugate for binding or tethering to the conductive surface. In one example, the aptamer conjugate provides suitable binding or tethering functional groups on one or both of the conductive surface and the aptamer, and can be coupled via one or more conjugation chemistries, such as click chemistry using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide / N-hydroxysulfosuccinimide (EDC / NHS) chemistry, silane-based conjugation using diazonium salt / thiolene click chemistry, phosphonate conjugation, (strept)avidin-biotin conjugation, silane conjugation, π-π stacking, bicyclo[6.1.0]nonyne (BCN)-azide conjugation, biotinylation, Cu(I)-catalyzed azide-alkyne click chemistry, etc. Aptamer conjugates can be attached or tethered to conductive surfaces using various methods, including CuAAC (CuAAC), tetrazine and alkene ligation (e.g., using trans-cyclooctene), and biocompatible strain-promoted azide-alkyne click chemistry (SPAAC) reagents, dibenzocyclooctyne (DBCO)-azide or DBCO-NHS reagents, DBCO-PEG-amine conjugation reagents, DBCO-PEG-maleimide conjugation reagents, DBCO-PEG-alcohol reagents, amine-reactive trans-cycloctene (TCO) reagents for tetrazine attachment, such as TCO-NHS ester, carboxyl / carbonyl-reactive TCO reagents (e.g., TCO amine or amine salt), TCO-PEG-DBCO reagents, etc. In other examples, polyethylene glycol (PEG) linkers are used.

[0172] In one example, at least a portion of the substrate 110 surface comprises carbon, graphene, graphene oxide, or carbon ink. In one example, at least a portion of the substrate surface is comprised of a nanomaterial. In one example, at least a portion of the substrate 110 surface comprises a carbon, graphene, or graphene oxide nanomaterial. In one example, the use of a carbon, graphene, or graphene oxide nanomaterial improves aptamer loading onto the substrate 110 surface to, among other things, optimize aptamer 102 loading and binding stability. In one example, aptamer conjugates can be prepared using a variety of methods, including diazonium salt / thiolene click chemistry, phosphonate conjugation, (strept)avidin-biotin conjugation, silane conjugation, π-π stacking, bicyclo[6.1.0]nonyne (BCN)-azide conjugation, biotinylation, Cu(I)-catalyzed azide-alkyne click chemistry (CuAAC), ligation of tetrazines and alkenes (e.g., using trans-cyclooctene), and biocompatible strain-promoted azide-alkyne click chemistry (SPAAC) reagents, dibenzofurans, and the like. These are configured for electrografting onto carbon-based electrodes using dicyclooctyne (DBCO)-azide or DBCO-NHS reagents, DBCO-PEG-amine conjugation reagents, DBCO-PEG-maleimide conjugation reagents, DBCO-PEG-alcohol reagents, amine-reactive trans-cyclooctene (TCO) reagents for tetrazine conjugation, such as TCO-NHS ester, carboxyl / carbonyl-reactive TCO reagents (e.g., TCO amine or amine salt), TCO-PEG-DBCO reagents, etc. Other examples include using polyethylene glycol (PEG) linkers for aptamer conjugates to increase nuclease resistance, or lipid conjugation to aptamers, or alternative nucleic acids for aptamer construction (e.g., L-DNA or L-RNA with increased -OH activity for binding / tethering), peptide nucleic acid (PNA) for aptamer construction, and combinations of the above.

[0173] In one example, the substrate 110 surface comprises gold, and at least a portion of the substrate 110 surface is configured to covalently bond, tether, or associate with an alkylthiol or mercaptothiol. In another example, at least a portion of the substrate 110 surface is configured to covalently bond a linear or branched aliphatic amine, a substituted or unsubstituted benzylamine, or a substituted or unsubstituted phenylamine, or a linear or branched aminoalkanoic acid, a substituted or unsubstituted aminobenzilic acid, or a substituted or unsubstituted aminophenylcarboxylic acid. In one example, at least a portion of the substrate 110 surface is chemically modified with streptavidin, avidin, gold, biotin, or a polymer such as dextrin and chitosan. In one example, a substrate comprising a gold surface is modified or treated with graphene oxide and / or zinc sulfide (ZnS) to improve binding or tethering of aptamers to the substrate.

[0174] In one example, at least a portion of the carbon, graphene, or graphene oxide nanomaterial substrate 110 surface comprises a covalently bonded linear or branched aliphatic amine, substituted or unsubstituted benzylamine, or substituted or unsubstituted phenylamine, or a covalently bonded linear or branched aminoalkanoic acid, substituted or unsubstituted aminobenzilic acid, or substituted or unsubstituted aminophenylcarboxylic acid. In one example, the linear or branched aliphatic amine, substituted or unsubstituted benzylamine, or substituted or unsubstituted phenylamine, or the covalently bonded linear or branched aminoalkanoic acid, substituted or unsubstituted aminobenzilic acid, or substituted or unsubstituted aminophenylcarboxylic acid is also covalently bonded to an aptamer 102 or aptamer-redox moiety 104 conjugate. For example, the substrate 110 surface is modified with a carboxylated material to enable covalent immobilization of amine-modified aptamers to exposed COOH groups via EDC / NHS chemistry.

[0175] Among exemplary nanomaterials, graphene oxide (GO) offers significant advantages for use in EAB devices due to its large surface area with multiple exposed carboxyl (COOH) and alcohol (COH) groups that can be used as anchor points for immobilizing aptamer-conjugated probes using a variety of different types of coupling chemistries. GO offers high versatility for functionalization and has demonstrated beneficial orientation effects in aptamer immobilization. Thus, in one example, a partially or fully embeddable sensor with a GO-functionalized substrate 110 surface (as a model carboxylated surface) serves as the working electrode for covalent immobilization of amine-functionalized aptamers. To covalently immobilize aptamer conjugates on the GO surface, for example, activation of the GO-carboxyl ("COOH") moiety can be performed via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide / N-hydroxysulfosuccinimide (EDC / NHS) chemistry, followed by the formation of corresponding amide bonds with amine groups present in the aptamer sequence. This immobilization strategy is expected to provide greater stability of the immobilized aptamer monolayer on the GO electrode surface, thus providing extended sensor lifetime and representing an alternative to thiol-based aptamer immobilization strategies. In a similar manner, activation of the GO-"COH" moiety with trialkoxysilane-modified aptamer conjugates can be performed. In one example, the aptamer conjugates can be electrochemically grafted onto the GO electrode surface.

[0176] In one embodiment, the substrate 110 surface is a carboxyl-functionalized substrate 110 surface, a thiol-functionalized substrate 110 surface, or a combination of a carboxyl-functionalized substrate 110 surface and a thiol-functionalized substrate 110 surface.

[0177] In one embodiment, the substrate 110 surface is essentially a carboxyl-functionalized substrate 110 surface. In one embodiment, the substrate 110 surface is essentially a carboxyl-functionalized substrate 110 surface that is substantially free of thiol functionalization.

[0178] In one example, the substrate 110 surface is essentially a GO-functionalized substrate 110 surface. In one example, the substrate 110 surface is essentially a GO-functionalized substrate 110 surface that is substantially free of thiol functionalization.

[0179] Aptamer / aptamer-signal transduction element conjugates In one example, one or more aptamer conjugates 102 of the AB or EAB of the present disclosure comprise an RNA or DNA nucleotide sequence. In one example, one or more aptamer conjugates 102 comprise at least one of a 2'-O-methyl modification of nucleotides, a disulfide bridge, a 3' cap with an inverted 2-deoxythymidine, a 3'-3'-thymidine linkage at the 3' end, a 2'-F modification, and a double-stranded section. In one example, one or more aptamer conjugates 102 comprise an RNA or DNA sequence having a first linker moiety at the 5' end and a reversible redox moiety at the 3' end. In one example, one or more aptamer conjugates 102 comprise an RNA or DNA sequence having a first linker moiety at the 3' end and a reversible redox moiety at the 5' end. In one example, a redox moiety, e.g., methylene blue, is attached to the oligo portion of the aptamer either internally in the sequence linked via a thymidine base, at the 5' end of the sequence, or at the 3' end via a modified thymidine or a 5-7 carbon spacer.

[0180] In one example, the first linker moiety on the 5' end of the aptamer 102 comprises an amino group, a carboxyl group, or a trialkoxysilane group. In one example, the first linker moiety of the aptamer 102 is physically or chemically bound to the substrate at the 5' end. In one example, the first linker moiety of the aptamer 102 is physically or chemically bound to the co-adsorbent at the 5' end. Alternatively, the first linker moiety on the 3' end of the aptamer 102 comprises an amino group, a carboxyl group, or a trialkoxysilane group, and the first linker moiety of the aptamer 102 is physically or chemically bound to the substrate at the 3' end. In one example, the first linker moiety of the aptamer 102 is physically or chemically bound to the co-adsorbent at the 3' end.

[0181] In one example, one or more aptamer conjugates 102 are neurotransmitter-binding aptamers. In one example, one or more aptamer conjugates 102 are dopamine- or glutamate-binding aptamers. In one example, one or more aptamer conjugates 102 are carbohydrate-, triglyceride-, or fatty acid-binding aptamers. In one example, one or more aptamer conjugates 102 are glucose-, glycerol-, or beta-hydroxybutyrate-binding aptamers. In one example, one or more aptamer conjugates 102 are glycopeptide antibiotic-binding aptamers. In one example, one or more aptamer conjugates 102 are vancomycin-binding aptamers. Combinations of different aptamer conjugates 102 on the same or different WE surfaces can be used to provide a multi-analyte monitoring EAB device.

[0182] In one embodiment, one or more aptamer conjugates are physically or chemically bound to a self-assembled monolayer (SAM). In one embodiment, one or more aptamer conjugates are physically or chemically bound to a mono- or polyfunctional alkanethiol or mercaptoalkanol. In one embodiment, one or more aptamer conjugates are physically or chemically bound to an alkylthiolbetaine. In one embodiment, one or more aptamer conjugates are physically or chemically bound to an aliphatic amine.

[0183] Aptamer Protection Layer (APL) It has been observed that certain attributes of the APL affect the suitability of the AB or EAB for sequential operation in vivo. For example, sequential ABs or EABs with an APL that has sufficient free volume for aptamer conformational changes, favorable ionic properties, sufficient porosity for analytes, and blocking of proteins, peptides, macrophages, and other immune response biologics positively impacts EAB performance. One or more of the above characteristics may further provide, directly or indirectly, stability of aptamer binding or tethering (reduced detachment from the substrate or SAM), reduced drift in signal or sensitivity over time in vivo, and extended in vivo performance compared to ABs or EABs without an APL.

[0184] In one example, the APL is a coating, matrix, membrane, domain, or layer. In another example, the APL is a coating, matrix, membrane, domain, or layer of a polymeric material. The polymeric material forming the basis of the APL can include one or more polymers, oligomers, coating layers, membranes, or matrices. In one example, the APL provides sufficient permeability to allow relevant analyte compounds to pass through it, e.g., to allow the analyte to pass through the membrane from the sample under test to reach the aptamer and enable transduction of a signal corresponding to the analyte concentration in the sample.

[0185] 3 is a schematic diagram of an exemplary APL according to the broadest aspect of the present disclosure. Thus, in one embodiment, APL 305 includes at least one polymer segment 302, 304. In one embodiment, the APL includes at least one polymer segment selected from the group consisting of polyurethanes, polyureas, poly(urethane ureas), epoxides, polyolefins, polysiloxanes, polyamides, polystyrenes, polyacrylates, polyethers, polyvinylpyridines, polyvinylpyrrolidones, polyesters, polycarbonates, and copolymers thereof.

[0186] The hydrophilicity of APL305 can be adjusted by the selection of the soft segments and soft segment ratios used during conventional PU or PUU synthesis. For example, soft segment components are shown in Figure 3 (hydrophilic and hydrophobic polyols). The hydrophobic soft segments can be PDMS, polycarbonate, polyester, polyether, or polymers with hydrophobic functional groups, such as fluorine or silicone. The hydrophobic segments can be present in the aforementioned APL at 1-50 wt%, 2-50 wt%, 5-50 wt%, 10-50 wt%, 15-50 wt%, 20-50 wt%, 25-50 wt%, 30-50 wt%, 10-20 wt%, or 15-25 wt%.

[0187] The hydrophilic soft segment can be polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, or zwitterionic polymer. By adjusting the chemical composition and / or molecular weight or distribution of the soft and hard segments in the PU or PUU, and / or by adding or removing functional groups, desired APL properties and functionality, such as surface charge / density and antifouling properties against proteins such as serum albumin (SA), can be achieved. The hydrophilic segment can be provided in the aforementioned APL at 1-50 wt%, 2-50 wt%, 5-50 wt%, 10-50 wt%, 15-50 wt%, 20-50 wt%, 25-50 wt%, 30-50 wt%, 10-20 wt%, or 15-25 wt%.

[0188] In one embodiment, the APL comprises a segmented multiblock polymer. Referring again to FIG. 3 , for example, the segmented multiblock polymer comprises soft segment 306 and hard segments (one or more of 308, 310, and 312). In one embodiment, the soft segment is hydrophobic or hydrophilic. In one embodiment, the soft segment is hydrophobic and hydrophilic. In one embodiment, the soft segment comprises a hydrophobic polyol and a hydrophilic polyol. In one embodiment, the APL comprises a segmented multiblock polyurethane polymer. In one embodiment, the APL comprises a segmented multiblock polyurethane, polyurethane-urea, or polyether-urethane, or polyether-urethane-urea polymer, copolymers, or blends thereof. In one embodiment, the hard segment comprises a urethane group, a urea group, or a combination thereof.

[0189] In one embodiment, the soft segment is one or more segments including polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof. In one embodiment, the soft segment is one or more segments including polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, polymers having repeating zwitterionic groups in their backbone and / or terminals (referred to herein as "zwitterionic repeating group polymers"), and blends or copolymers thereof. In one embodiment, end group functionalized-polyurethane (EGFPU) or polyurethane urea (EGFPUU) polymers can be used. EGFU / EGFPUU can be synthesized using reactive functional monomers / oligomers that end-capping polyurethane reaction intermediates to form polyurethanes with functional groups at one or both chain ends. The functional groups can be further deprotected to form reactive thiol groups that connect to the substrate 110 surface, e.g., gold.

[0190] Polyurethane and polyurethane-urea polymers can be produced by the condensation reaction of a diisocyanate with a difunctional hydroxyl-containing material or a difunctional amine-containing material. Polyurethane-ureas are polymers produced by the condensation reaction of a diisocyanate with a difunctional amine-containing material. In some examples, the diisocyanate includes an aliphatic diisocyanate containing from about 4 to about 8 methylene units. Diisocyanates containing alicyclic moieties can also be useful in preparing the polymer and copolymer components of the membranes of the present disclosure.

[0191] In one embodiment, end-group functionalized polyurethane (EGFPU) or polyurethane urea (EGFPUU) polymers can be used, as disclosed, for example, in commonly assigned U.S. Patent No. 10,413,227 (B2). EGFU / EGFPUU can be synthesized using reactive functional monomers / oligomers that end-capping polyurethane reaction intermediates to form polyurethanes with functional groups at one or both chain ends. The functional groups can be further deprotected to form reactive thiol groups that attach to the substrate 110 surface, e.g., gold.

[0192] For example, an exemplary APL hydrophobic-hydrophilic segmented copolymer component is a polyurethane polymer containing approximately 20% hydrophilic polyethylene oxide. The polyethylene oxide portion of the copolymer is thermodynamically driven to separate from the hydrophobic portion of the copolymer and the hydrophobic polymer component. In one example, it has been observed that approximately 20% of the polyethylene oxide-based soft segment portion of the copolymer used to form the APL affects the water absorption rate and subsequent analyte permeability of the APL membrane. In one example, the exemplary APL is prepared as an aqueous dispersion for use with the aptamer-signal transduction element conjugate. For example, a betaine-functionalized hydrophilic aliphatic polyurethane can be prepared as an aqueous dispersion that can be combined with an aqueous solution of the aptamer-signal transduction element conjugate.

[0193] Incorporation of zwitterionic repeat units into the aforementioned polyurethanes, polyurethane-urea polymers, polymers can be achieved by using zwitterionic monomers that have or can be bonded to a diol or diamine. Examples of such zwitterionic monomers include:

[0194] [ka] wherein X is one or two of -OH, -NHR, -NH, or -SH; W, Y, and Z are independently branched or straight-chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, any of which may be optionally substituted with O, OH, halogen, amido, or alkoxyl; R is H, branched or unbranched acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; and R, R, and R are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl.

[0195] These compounds can be reacted with diisocyanates to form polyurethanes or polyureas containing zwitterionic repeating units. Alternatively, the carboxylate, sulfonate, phosphinate, or phosphonate moieties of the precursor zwitterionic repeating units can be protected, and then the protecting groups can be removed after polymerization. In another alternative, the amine can be a tertiary amine that is quaternized by alkylation after polymerization. Further examples of PU and PUU polymers with zwitterionic repeating units can be found in commonly assigned U.S. Patent Application Publication No. 20170188923, U.S. Patent No. 11112377, and U.S. Patent No. 11179079, the disclosures of which regarding such polymers and their synthesis are incorporated herein by reference.

[0196] In one embodiment, the APL is composed of a non-polyurethane polymer. Examples of materials that can be used to make non-polyurethane-type APLs include vinyl polymers, polyethers, polyesters, polyamides, polysilicones, poly(dialkylsiloxanes), poly(alkylarylsiloxanes), poly(diarylsiloxanes), polycarbosiloxanes, polycarbonates, Nafion (sulfonated tetrafluoroethylene), natural polymers such as cellulose and protein-based materials, as well as blends, copolymers, or combinations thereof with or without the aforementioned polyurethanes or polyether-urethane-urea polymers.

[0197] The APLs disclosed herein can be formulated into a mixture that can be drawn into a film or applied to a surface using any method known in the art (e.g., spraying, painting, dip coating, vapor deposition, molding, 3D printing, lithographic techniques (e.g., photolithography), micro- and nano-pipetting printing techniques, silk screen printing, etc.). The mixture can then be cured under elevated temperatures (e.g., 50-150°C). Other suitable curing methods can include, for example, ultraviolet light or gamma radiation.

[0198] In one example, the aptamer protection layer is at least partially crosslinked using a crosslinker in an amount sufficient to crosslink the APL without inactivating the aptamer or substantially reducing the ability of the aptamer present therein to undergo a conformational change sufficient to provide signal transduction, hi one example, the aptamer protection layer is fully crosslinked using a crosslinker in an amount sufficient to crosslink the APL without substantially reducing aptamer signal transduction.

[0199] Suitable crosslinkers include isocyanates, carbodiimides, glutaraldehyde or other aldehydes, aziridines, silanes, epoxies, acrylates, free radical-based agents, ethylene glycol diglycidyl ether (EGDE), poly(ethylene glycol) diglycidyl ether (PEGDE), dicumyl peroxide (DCP), PVP-PEGDE, or PVP-PEG. In one embodiment, about 0.1% to about 15% w / w of crosslinker is added based on the total dry weight of crosslinker and polymer added when blending the components (in one example, about 1% to about 10%). During the curing process, it is believed that substantially all of the crosslinker reacts, leaving substantially no detectable unreacted crosslinker in the final film.

[0200] In one embodiment, the APL is a conductive polymer. In one embodiment, the APL is a functionalized polymer. The APL can be functionalized with, for example, 1-50 wt%, 2-50 wt%, 5-50 wt%, 10-50 wt%, 15-50 wt%, 20-50 wt%, 25-50 wt%, 30-50 wt%, 10-20 wt%, or 15-25 wt% of a functional moiety. The functionalized polymer can be configured to bond with a substrate, a SAM, or another layer, film, matrix, region, or polymer.

[0201] In one embodiment, the functionalized polymer comprises alkanethiol groups, hi one embodiment, the alkanethiol groups are present at the ends of the functionalized polymer chain or the alkanethiol groups are present along the backbone of the functionalized polymer chain.

[0202] In one embodiment, the functionalized polymer comprises mercaptoalkanol groups, hi one embodiment, the mercaptoalkanol groups are present at the ends of the functionalized polymer chain or along the backbone of the functionalized polymer chain.

[0203] In one embodiment, the APL comprises a zwitterionic group compound or a zwitterionic repeating group compound. In one embodiment, the functionalized polymer is prepared using at least one of the following polymerizable zwitterionic monomer structures:

[0204] [ka] wherein X is O, NH, or NR4; Y and Z are independently branched or straight-chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, which may be optionally substituted with OH, halogen, or alkoxyl; and R1, R3, R4, and R5 are independently H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl.

[0205] For example, APLs can include zwitterionic monomers including N-(2-methacryloyloxy)ethyl-N,N-dimethylammoniopropanesulfonate, N-(3-methacryloylimino)propyl-N,N-dimethylammoniopropanesulfonate, 2-(methacryloyloxy)ethylphosphatidylcholine, and 3-(2′-vinyl-pyridinio)propanesulfonate, alone or in combination with other polymer structures / backbones.

[0206] In one example, the APL of the present disclosure provides a zwitterionic repeat group compound in an amount capable of adjusting or maintaining the ionic strength or pH of an aptamer and / or a transducer (e.g., a redox moiety) and / or a substrate. In one example, one or more zwitterionic repeat groups comprise a betaine compound or a derivative thereof. In one example, the zwitterionic repeat groups are present at the termini of a functionalized polymer chain or along the backbone of a functionalized polymer chain.

[0207] In one embodiment, the functionalized polymer comprises an alkanethiol group and a zwitterionic repeating group. In one embodiment, the functionalized polymer comprises an alkanethiol and a betaine group. In one embodiment, the functionalized polymer comprises a mercaptoalkanol group and a zwitterionic repeating group. In one embodiment, the functionalized polymer comprises a mercaptoalkanol group and a betaine group. In one embodiment, the functionalized polymer comprises an arylthiol and a zwitterionic repeating group. In one embodiment, the functionalized polymer comprises an arylthiol and a betaine group. In one embodiment, the functionalized polymer comprises an arylmercaptoalkanol group and a zwitterionic repeating group. In one embodiment, the functionalized polymer comprises an arylmercaptoalkanol group and a betaine group. In one embodiment, the functionalized polymer comprises a benzylthiol and a zwitterionic repeating group. In one embodiment, the functionalized polymer comprises a benzylthiol and a betaine group. In one embodiment, the functionalized polymer comprises a benzylmercaptoalkanol group and a zwitterionic repeating group. In one embodiment, the functionalized polymer comprises benzylmercaptoalkanol groups and betaine groups.

[0208] In one example, the APL is physically or chemically bound to at least a portion of the substrate surface. In one example, the APL is physically or chemically bound to at least a portion of the substrate surface and one or more aptamer conjugates are physically or chemically bound to at least a portion of the substrate surface. In one example, the APL is physically or chemically bound to at least a portion of the substrate surface and one or more aptamer conjugates are physically or chemically bound to at least a portion of the substrate surface, and a substantial remainder of the substrate surface further comprises a physically or chemically bound co-adsorbate.

[0209] In one example, the APL provides sufficient free volume to allow for reversible conformational changes of the one or more aptamer conjugates, hi one example, at least one of the one or more aptamer conjugates is physically or chemically bound to an aminoalkanoic acid.

[0210] In one example, one or more aptamer conjugates may be present, e.g., 10 -9 , 10 -10 , 10 -11 , 10 -12 ~10 -13 molecules / cm 2 The aptamers present on the substrate surface are present at a density of 0.01 to 0.01 . Other densities can be used. In one example, the aptamers present on the substrate surface have substantially similar architectures (deviation of less than 2 base pairs), the same transducer or redox moiety, the same conjugate linkage chemistry linking the aptamer to the working electrode surface, SAM, or co-adsorbate, the same aptamer / co-adsorbate mass ratio and / or density, and the same manufacturing history. In one example, the aptamers present on the substrate surface have different architectures (deviation of more than 2 base pairs), different or the same transducer or redox moiety, different or the same conjugate linkage chemistry linking the aptamer to the working electrode surface, SAM, or co-adsorbate, different aptamer / co-adsorbate mass ratio and / or density, and different or the same manufacturing history.

[0211] signal conversion element In one embodiment, the signal transduction element includes a redox moiety. The redox moiety may include any compound that causes a change in electron transfer rate upon change in its proximity to an electrode at a bias potential. Exemplary redox species include methylene blue, organometallic redox moieties, ferrocene, viologens, anthraquinones or any other quinones, ethidium bromide, daunomycin, metalloporphyrin complexes, crown ether metal complexes, bis-pyridine metal complexes, bis-imidazole metal complexes, tris-pyridine metal complexes, ethylenetetracetic acid (EDTA)-metal complexes, and cytochromes. In one embodiment, the reversible redox moiety includes iron, iridium, ruthenium, osmium, a thiazine dye, or a derivative thereof. In one embodiment, the reversible redox moiety includes ferrocene or methylene blue.

[0212] In one embodiment, the sensor is configured for continuous, semi-continuous, sequential, or random signal acquisition. In one embodiment, the sensor is configured for percutaneous insertion.

[0213] Co-adsorbent In one embodiment, the disclosed AB or EAB device includes one or more co-adsorbents. The co-adsorbents coat the substrate (adsorbent) and function to alter the substrate's response to exposure to the ambient environment, thereby eliminating or reducing undesired activity or reactions. The effectiveness of the co-adsorbents can be measured experimentally, for example, by tracking baseline current levels when the substrate is biased with an electrical potential. In one embodiment, the co-adsorbents are configured to independently provide a surface energy-modulating environment, a phase separation-modulating environment, and / or a molecular interaction-modulating environment between the co-adsorbate molecules and / or the co-adsorbate and aptamer molecules, the aptamer 102 and the substrate 110 surface, any monolayer 103, and / or the aptamer-protecting material 105.

[0214] In one example, one or more co-adsorbents independently provide ionic strength, and the one or more co-adsorbents are present in an amount capable of adjusting or maintaining the ionic strength in proximity to at least one aptamer conjugate and / or the substrate surface.

[0215] In one embodiment, at least a portion of the substrate 110 surface further comprises one or more co-adsorbents. In one embodiment, the one or more co-adsorbents independently comprise multiple functional groups. Figures 4A and 4B show schematic diagrams of exemplary co-adsorbents 402a and 402b, respectively, according to the broadest aspects of the present disclosure. Accordingly, Figure 4A shows an enlarged cross-sectional schematic diagram of the substrate 110WE surface (as a working electrode) of an embedded EAB with an exemplary architecture of co-adsorbent 402a having linear segments 406a and end-group segments 404a. Figure 4B shows an enlarged cross-sectional schematic diagram of the substrate 110WE surface (as a working electrode) of an embedded EAB with an exemplary architecture of co-adsorbent 402a having linear segments 406b and backbone segments 404b. The linear segments 406a, 406b can include, for example, alkyl, alkylthiol, phenylthiol, benzylthiol, arylthiol, mercaptoacanol, alkylsilane, aromatic silane, or alkylaromatic silane, as disclosed herein. In one example, the linear segments 406a, 406b include aromatic thiol or alkylaromatic thiol. In one example, the end group segment 404a or the backbone segment 404b can be a zwitterionic group or a repeating zwitterionic group, as disclosed herein. The substrate 110 can include random and / or patterned combinations of co-adsorbents 402a and 402b at various substrate surface area ratios. The co-adsorbents 402a, 402b can be attached to the substrate 110 surface (indicated by "X" in FIGS. 4A and 4B) in various ways, e.g., thiol, amine, amino, carboxyl, carboxylamine, or carboxylamino, via EDC / NHS chemistry, e.g., click chemistry, as discussed herein. FIG. 4C shows an enlarged cross-sectional schematic diagram of the substrate 110WE surface (as the working electrode) of an embedded EAB having an exemplary architecture of co-adsorbent 403 having linear segments 406 and linear sections 405, where linear segments 406 can be, for example, alkyl, alkylthiol, mercaptoacanol as disclosed herein, and linear sections 405 can be zwitterionic groups or repeating zwitterionic groups as disclosed herein.The co-adsorbents 403 can be attached to the substrate 110 surface in a variety of ways, e.g., thiol, amine, amino, carboxyl, carboxylamine, or carboxylamino, for example, via EDC / NHS chemistry or click chemistry as discussed herein. The substrate 110 surface can include random and / or patterned combinations of co-adsorbents 402a, 402b, and 403 at various substrate surface area ratios.

[0216] In one embodiment, the continuously monitored AB or EAB of the present disclosure includes one or more co-adsorbents associated with the substrate 110 surface, where the one or more co-adsorbents are chemically distinct.

[0217] In one example, the functionalized APL can also function, in part, as a co-adsorbent. Thus, in one example, at least a portion of the substrate surface comprises a functionalized APL, at least a portion of the substrate surface comprises one or more co-adsorbents, and at least a portion of the remaining portion of the substrate surface comprises one or more aptamer conjugates, and the total percentage of the substrate surface and the remaining portion can be 100 percent or less.

[0218] In one embodiment, at least a portion of the substrate surface, the one or more co-adsorbents, and a portion of the remainder of the substrate surface comprise one or more aptamer conjugates. In one embodiment, the remainder of the substrate surface is about 50% of the total surface area of ​​the substrate. In one embodiment, the remainder of the substrate surface is less than 50% but greater than 0% of the total surface area of ​​the substrate. In one embodiment, the remainder of the substrate surface is greater than 50% but less than 100% of the total surface area of ​​the substrate.

[0219] In one example, at least a portion of the substrate surface comprises one or more co-adsorbents, and a portion of the remaining portion of the substrate surface comprises one or more aptamer conjugates physically or chemically bound to the substrate. In one example, one or more co-adsorbents are physically or chemically bound to the substrate surface, and a portion of the remaining portion of the substrate surface comprises one or more aptamer conjugates physically or chemically bound to at least a portion of the co-adsorbents.

[0220] In one embodiment, the co-adsorbate comprises a self-assembled monolayer (SAM). In one embodiment, the co-adsorbate comprises a mono- or polyfunctional alkanethiol.

[0221] In one embodiment, the thiol functional group of the monofunctional alkanethiol or polyfunctional alkanethiol is covalently bonded to at least a portion of the surface of the substrate. In one embodiment, the thiol functional group of the monofunctional alkanethiol or polyfunctional alkanethiol is covalently bonded to the surface of a gold substrate.

[0222] In one embodiment, the co-adsorbent comprises a mono- or poly-functional mercaptoalkanol. In one embodiment, the thiol functional group of the mono- or poly-functional mercaptoalkanol is covalently bonded to at least a portion of the surface of the substrate. In one embodiment, the thiol functional group of the mono- or poly-functional mercaptoalkanol is covalently bonded to at least a portion of the surface of the gold substrate.

[0223] In one embodiment, the co-adsorbate comprises a zwitterionic repeat group associated with at least a portion of the substrate surface. In one embodiment, the co-adsorbate comprises a zwitterionic repeat group bound to at least a portion of the substrate surface. In one embodiment, the co-adsorbate comprises a zwitterionic repeat group covalently attached to at least a portion of the substrate surface. In one embodiment, the zwitterionic repeat group comprises a betaine group, e.g., a sulfobetaine group or a carboxybetaine group.

[0224] In one embodiment, the zwitterionic repeating group comprises an ammoniophosphate or lecithin analog, an ammoniophosphonate, an ammoniophosphinate, an ammoniosulfonate, an ammoniosulfate, an ammoniocarboxylate, or a combination thereof.

[0225] In one embodiment, the zwitterionic repeating group comprises an alkanethiol betaine. In one embodiment, the alkanethiol is linear and comprises multiple betaine groups along its chain. In one embodiment, the alkanethiol is a terminally terminated mono- or dithiol having at least one betaine group along its chain. In one embodiment, the alkanethiol is linear and comprises a terminally terminated betaine group. In one embodiment, the thiol group of the terminally terminated dithiol alkanethiol is covalently bonded to the substrate surface.

[0226] In one embodiment, the zwitterionic repeating group comprises a mercaptoalkanol betaine. In one embodiment, the mercaptoalkanol is linear and comprises multiple betaine groups along the chain. In one embodiment, the mercaptoalkanol is linear and comprises terminal terminating betaine groups. In one embodiment, the thiol group of the mercaptoalkanol is covalently bonded to the substrate surface.

[0227] In one embodiment, the co-adsorbate has the following structure:

[0228] [ka] (In the formula,

[0229] [ka] represents a hydrocarbon chain, the zwitterionic units are attached to the backbone and the charges are on side groups pendant to the backbone, or the zwitterionic units are such that one or both charges are on the backbone, R1 and R2 are independently branched or unbranched acyclic alkyl, substituted or unsubstituted cyclic alkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocyclic, X is -OH, -NHR1, -NH2, or -SH, and n is an integer from 2 to about 1000; or

[0230] [ka] wherein X is -OH, -NHR, -NH, or -SH; W, Y, and Z are independently branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, any of which may be optionally substituted with O, OH, halogen, amido, or alkoxyl; R is H, branched or unbranched acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; and R, R, and R are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl.

[0231] In one embodiment, the co-adsorbate has one or more of the following structures:

[0232] [ka] wherein R1 is H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R2, R3, and R4 are independently selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; and n is an integer from 2 to 24.

[0233] In one embodiment, the co-adsorbent is ammoniosulfonate (sulfobetaine) or ammoniosulfate, structure:

[0234] [ka] wherein Z is a branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R is H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R and R are independently selected from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and one or more of R, R, R, and Z are substituted with a polymerizable group, and the structure:

[0235] [ka] wherein Z is a branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R1 is H, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R2 and R3 are independently selected from alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and one or more of R1, R2, R3, and Z are substituted with a polymerizable group.

[0236] In each of these monomers, Z can have a length of 1 to 12 atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 atoms, and any of these values ​​can form the upper or lower end of a range.

[0237] These compounds or monomers can be prepared by methods known to those skilled in the art, such as those detailed in Laschewsky, "Structures and synthesis of zwitterionic polymers," Polymers 6:1544-1601, 2014. In certain embodiments, the disclosed polyzwitterions can have repeating zwitterionic units derived from any of the zwitterionic compounds or monomers disclosed above. Exemplary zwitterionic compounds include octamidopropyl betaine, oleamidopropyl betaine, octyl sulfobetaine, caprylyl sulfobetaine, lauryl sulfobetaine, myristyl sulfobetaine, palmityl sulfobetaine, stearyl sulfobetaine, betaine (trimethylglycine), octyl betaine, phosphatidylcholine, glycine betaine, poly(carboxybetaine), poly(sulfobetaine), and derivatives thereof. Exemplary monomers comprising one or more pendant or terminal groups with zwitterionic groups include or are derived from octamidopropyl betaine, oleamidopropyl betaine, octyl sulfobetaine, caprylyl sulfobetaine, lauryl sulfobetaine, myristyl sulfobetaine, palmityl sulfobetaine, stearyl sulfobetaine, betaine (trimethylglycine), octyl betaine, phosphatidylcholine, glycine betaine, poly(carboxybetaine), and poly(sulfobetaine).

[0238] In one embodiment, the co-adsorbate is a terminally terminated mono- or dithiol having at least one zwitterionic group or zwitterionic repeating group, as disclosed herein. In one embodiment, controlling the ionic strength, pH, etc., includes configuring the APL backbone or one or more appendages from the backbone with one or more zwitterionic betaine groups.

[0239] In one embodiment, the APL comprises an alkanethiol or (aryl)mercaptoalkanol and one or more zwitterionic groups. In one embodiment, controlling the ionic strength, pH, etc. comprises providing the APL in combination with a mercaptoalkanol having a zwitterionic betaine group.

[0240] method The APL-EAB construct of the present disclosure provides advantages over EABs that do not have APL. For example, the APL-EAB construct of the present disclosure can be used in a method for determining the in vivo concentration of an analyte. For example, the method can include contacting a biological fluid containing an analyte with the APL-EAB construct of the present disclosure in vivo, wherein the EAB is bound to a conductive substrate, the EAB is encapsulated in the APL, the APL is permeable to the analyte, and the EAB generates a signal upon interaction with the analyte.

[0241] The APL-EAB constructs of the present disclosure are configured to receive a bias voltage that varies to reversibly oxidize and reduce a redox probe associated with an aptamer, the aptamer being associated with a conductive substrate surface. The APL-EAB constructs of the present disclosure can be used in methods that include interrogating a conductive substrate or an APL-aptamer-redox moiety conjugate. The method can further include detecting a signal generated by the aptamer-redox moiety conjugate in the presence of a concentration of an analyte, and correlating the in vivo concentration of the analyte based on the detected signal, a change in the signal, a difference in the signal, or the like. In one example, signal transduction by the APL-EAB constructs of the present disclosure is determined by the rate of electron transfer from the reversible redox probe, and the difference in the electron transfer rate correlates with the analyte concentration.

[0242] In one embodiment, the interrogating is continuous, semi-continuous, sequential, or random temporal detection of the signal. In one embodiment, the method can further include adjusting the signal based on a background signal generated as a result of non-specific binding of the aptamer biosensor to generate an adjusted signal. Two or more working electrodes, with or without the APL-EAB construct of the present disclosure, can be used. The method can further include determining the in vivo concentration of the analyte over a period of time based on the adjusted signal.

[0243] In one embodiment, investigating includes a differential measurement technique. Exemplary differential measurement techniques include, for example, investigating at a first square wave voltammetry (SWV) frequency to obtain a first signal and at a second SWV frequency to obtain a second signal, taking the difference between the two signals, and dividing by the average of the two signals to obtain an adjusted signal. In one embodiment, investigating includes chronoamperometry. In one embodiment, investigating includes cyclic voltammetry.

[0244] In one example, the APL of the present disclosure can control or regulate the intermolecular interaction between the aptamer conjugate and the APL. In another example, the APL construct of the present disclosure, alone or in combination with the coadsorbent of the present disclosure, results in reduced aptamer detachment from the substrate surface. In another example, the APL of the present disclosure can control or regulate the diffusion of the aptamer from the vicinity around the substrate surface. Thus, when reversible desorption / detachment of the aptamer from the substrate occurs, the APL of the present disclosure can maintain the aptamer in proximity to the substrate surface to increase aptamer resorption / rebinding. In one example, the APL of the present disclosure is partially crosslinked. The APL of the present disclosure can be crosslinked in the presence of an aptamer-converting moiety with little or no adverse effect on APL-EAB performance, as discussed below.

[0245] In one embodiment, the APL of the present disclosure can be used to extend the in vivo end-of-life of an EAB device. For example, the APL of the present disclosure has demonstrated an extended end-of-life of up to 20 hours in bovine serum albumin. It is contemplated that the APL of the present disclosure can provide an EAB with an in vivo end-of-life performance of up to 1 day, 2 days, 1 week, 2 weeks, 3 weeks, or 1 month.

[0246] In one example, the APLs of the present disclosure, alone or in combination with a SAM or co-adsorbent, provide a method for controlling or adjusting the ionic strength of an aptamer-signal transduction element conjugate. For example, a functionalized APL of the present disclosure, e.g., a betaine-functionalized APL in combination with one or more co-adsorbents, can be present in an amount that can adjust or maintain the ionic strength of the aptamer-signal transduction element conjugate.

[0247] A method for fabricating an APL-EAB device of the present disclosure includes presenting an aptamer containing a reversible redox moiety on the surface of a conductive substrate, and presenting an APL on a portion of the surface of the conductive substrate to encapsulate the aptamer conjugate in the APL. Alternatively, an APL-EAB of the present disclosure is combined with an aptamer containing a reversible redox moiety and presented on the substrate surface.

[0248] Experimental results A series of exemplary APLs were developed and tested with aptamer-redox moiety conjugate EAB constructs to evaluate the effectiveness of the APLs in providing improvement of one or more attributes of the constructs. Characteristics of a representative sampling of APLs are summarized in Table 1.

[0249] Table 1. Exemplary APLs. PUU = aliphatic polyurethane urea segmented block copolymer. PU = aliphatic polyurethane segmented block copolymer. Hydrophilic segment = polyethylene glycol and polycarbonate. Hydrophobic segment = polydimethylsiloxane. Functional content = sulfobetaine or carboxybetaine. Crosslinker = polyglycol polyglycidyl (PEG-PG). Weight percent values ​​may vary + / - 10%. [Table 1]

[0250] Figures 5A and 5B are representative graphs of experimental voltammetry readout versus electron transfer readout (charge versus frequency) data for an exemplary aptamer biosensor with and without an APL. In this example, the co-adsorbate was 6-mercapto-1-hexanol, the conjugation chemistry was thiol association on the gold substrate, and the APL used was PUU-3.

[0251] The aptamers tested were specific for vancomycin and aminoglycosides. Figures 5A and 5B demonstrate a kinetic differential measurement (KDM) signal of approximately 71% at a 50 µmol / L analyte spike in pre-serum PBS buffer solution without APL ("control EAB"), while the APL-coated sample ("APL-EAB") provides a KDM signal of 67% under the same conditions.

[0252] Referring to Figures 6A and 6B, samples after 20 hours of exposure to biological fluids (50 μm serum incubation) demonstrate that the APL sample PUU-9 maintains good KDM, while the control shows significant degradation of KDM. The APL sample demonstrates approximately four times the signal of the control after 20 hours.

[0253] Figures 7A and 7B are representative graphs of experimental charge vs. frequency data for protein fouling of a control versus the same aptamer conjugate with aptamer-protected material. The data demonstrate the benefit of APL in maintaining EAB response by resisting biofouling compared to the uncoated control.

[0254] Figures 8A and 8B are representative graphs of experimental current versus frequency voltammogram data taken at different time intervals for an exemplary aminoglycoside aptamer biosensor in protein-spiked buffer without aptamer-protecting material compared to an APL-aminoglycoside aptamer sample, respectively. The data in Figure 8A show that the uncoated EAB has a continuous decay in signal output in a protein-containing environment, e.g., a gradual shrinkage in peak height over time, while in contrast, the APL-aminoglycoside aptamer sample in Figure 8B shows minimal change in readout.

[0255] Figure 9A is a representative graph of experimental normalized readout percentage versus time showing drift of control EABs compared to APL EABs without a biofouling challenge. Samples were exposed to bovine serum albumin (BSA), and as shown, the control EABs without APL began to drift from biofouling after less than 1 hour, while the APL-EABs provided stable performance for at least 5 hours.

[0256] Figure 9B is a representative graph of experimental normalized readout percentage versus time, showing the drift of control EAB compared to APL-EAB in a buffer solution containing biofouling proteins. Samples were exposed to bovine serum albumin (BSA). As shown, the control EAB without APL began to drift from biofouling after less than 1 hour, whereas APL-EAB, e.g., sample PUU-9, provided stable performance for at least 22 hours (without analyte challenge). Thus, a zwitterionic functional content of at least 10 wt% in PU or PUU results in one or more performance improvements of the EAB, such as calibration stability, storage stability, drift stability, localized pH stability, and interference reduction.

[0257] Figure 9C is a representative graph of experimental normalized readout percentage versus time illustrating the stability of the exemplary APL EAB sensor PUU-3 in PBS at 37°C. As shown, the APL-EAB sample exhibited stability for at least 6 days, with at least 80% signal remaining. This data demonstrates the stability-enhancing properties of the APLs of the present disclosure.

[0258] 10A and 10B are representative graphs of experimental current versus frequency voltammogram data for an exemplary vancomycin aptamer biosensor without aptamer protection material compared to an APL-vancomycin aptamer sample, respectively. Again, the data show that while the uncoated EAB has a continuous decay in signal output during potential cycling, the APL-vancomycin aptamer sample is significantly more robust.

[0259] 11, a representative graph of experimental sensor response percentage versus analyte concentration is shown for exemplary EABs with and without aptamer-protecting material exposed to various analyte concentrations. The vancomycin EAB with APL (PUU-3) provided essentially equivalent signal-concentration curves to the vancomycin EAB without APL, although the sensor response was slightly lower at a given vancomycin concentration.

[0260] In the aforementioned experiments, the substrate used was a standard gold disk electrode. Follow-up studies using other electrode shape factors showed that the EAB-APL is not substrate-dependent and can be reproduced using, for example, wire electrode shape factors. The electrode shape factor affects the absolute signal level, at least due to differences in surface area.

[0261] Figures 12A and 12B are representative calibration graphs of an exemplary vancomycin aptamer biosensor with different co-adsorbents, 6-mercapto-1-hexanol (MCH) and 8-mercapto-1-hexanol (MCO), challenged with analyte concentrations of 0 μM, 10 μM, and 30 μM, respectively. The data from Figures 12A and 12B demonstrate successful calibration and the compatibility of various co-adsorbents with the APL of the present disclosure.

[0262] Figures 13A and 13B are representative graphs of the shelf-life performance of uncoated versus APL-coated EABs, respectively, after 5 hours of ambient storage. The uncoated sensors showed significant performance degradation after storage, accompanied by a large background current, while minimal change in performance was observed for the APL-EABs.

[0263] Figures 13C and 13D are representative graphs of calibration and drift data for an exemplary APL-EAB (targeting vancomycin) after one month of storage in an ambient air environment at room temperature and relative humidity in the dark. Figures 13E and 13F are representative graphs of calibration and drift data for an exemplary APL-EAB (targeting vancomycin) after two months of storage in an ambient air environment at room temperature and relative humidity in the dark. The data from Figures 13C-13F demonstrate good calibration and drift performance over at least two months using the disclosed APL and APL with a co-adsorbent.

[0264] Drug-releasing layer Devices and probes inserted or implanted percutaneously into subcutaneous tissue traditionally induce a foreign body response (FBR), which involves the invasion of inflammatory cells that ultimately form a foreign body capsule (FBC) as part of the body's response to the introduction of a foreign body. Continuous monitoring systems discussed herein include continuous analyte monitoring systems configured to simultaneously, sequentially, and / or randomly monitor one, two, or more analytes (encompassing events that can occur independently on the order of picoseconds, nanoseconds, milliseconds, seconds, or minutes) to predict health-related events and health system performance (e.g., current and future performance of a body system, such as a cardiovascular, respiratory, gastrointestinal, or other system, or combination of organs or systems). In embodiments, the insertion or implantation of a device, e.g., an EAB sensing device, can result in an acute inflammatory response that resolves into chronic inflammation with the concomitant construction of fibrous tissue, as described in detail above. Eventually, over a period of time, a mature FBC, primarily comprising contractile fibrous tissue, forms around the device. See Shanker and Greisler, Inflammation and Biomaterials in Greco RS, ed., "Implantation Biology: The Host Response and Biomedical Devices," pp. 68-80, CRC Press (1994). FBCs surrounding conventional implanted devices have been shown to impede or block analyte transport across the device-tissue interface. Thus, continuous, extended-life analyte transport in vivo (e.g., beyond the first few days) has traditionally been considered unreliable or impossible.

[0265] In some embodiments, certain aspects of the FBR during the first few days may play a role in noise. Some sensors have been observed to function more poorly during the first few hours after insertion than they do later. This is exemplified by noise and / or suppression of the signal during the first few hours after insertion (e.g., about 2 to about 24 hours). These abnormalities often resolve naturally, after which the sensor becomes less noisy, more sensitive, and more accurate than initially. Some transcutaneous and fully implantable sensors have been observed to experience noise for a period of time after application to a host (i.e., inserted percutaneously or fully implanted under the skin).

[0266] 2D , a drug-release layer, membrane, matrix, or coating 113 can be positioned adjacent to or directly adjacent to the APL 105. In one example, the AB or EAB continuous sensor of the present disclosure includes an immune response-attenuating or drug-release layer configured to interact with the host's immune system or release an active agent into the sensor's environment. In one example, the immune response-attenuating layer includes an active agent bound to or entrapped within the layer, such as a covalently bound active agent (e.g., a dexamethasone derivative or analog) or a surface-exposed active agent (e.g., silver nanoparticles). In one example, the drug-release layer includes an active agent configured to release from the layer over time to mitigate or attenuate an immune response. Such drug-release layers include, for example, segmented polyurethane polymers containing dexamethasone and / or dexamethasone acetate and / or other dexamethasone derivatives or analogs, as disclosed in commonly assigned U.S. Patent Application Publication No. 17 / 945,585, incorporated herein by reference.

[0267] manufacturing The substrate can be formed by a variety of fabrication techniques (e.g., bulk metal processing, deposition of metal on a substrate, etc.). In one example, the substrate is a plated wire (e.g., platinum on a steel wire) or bulk metal (e.g., gold wire). EAB substrates formed from bulk metal wire (e.g., as opposed to deposited electrodes) are believed to offer superior performance, including increased assay stability, simplified manufacturability, resistance to contamination (e.g., that may be introduced in the deposition process), and improved surface reactions without peeling or delamination (e.g., due to material purity). The substrate can be a metal wire with an outer insulator. The substrate can be multiple metal wires, each with an outer insulator.

[0268] In embodiments in which an outer insulator is disposed around the substrate, portions of the coated assembly structure may be stripped or otherwise removed, for example, by hand, excimer laser, chemical etching, laser ablation, grit blasting (e.g., with sodium bicarbonate, solid carbon dioxide, or other suitable grit), etc., to expose the electrochemically active surface. Alternatively, portions of the electrode may be masked before depositing the insulator to maintain exposed electrochemically active surface area. In one exemplary embodiment, grit blasting is preferably implemented to expose the electrochemically active surface, utilizing a grit material that is hard enough to ablate the polymer material but soft enough to minimize or avoid damage to the underlying metal electrode (e.g., platinum electrode). While various "grit" materials (e.g., sand, talc, walnut shells, crushed plastic, sea salt, solid carbon dioxide, etc.) can be used, in some embodiments, sodium bicarbonate is an advantageous grit material because it is hard enough to ablate, for example, the parylene coating without damaging the underlying platinum conductor. One additional advantage of sodium bicarbonate blasting includes its abrasive action on metal as it strips the polymer layer, thereby eliminating a cleaning step that might otherwise be required. Etching (e.g., chemical or plasma) or other methods can be used to provide nanopores and / or micropores in the substrate surface.

[0269] In some embodiments, a radial window is formed through the insulating material to expose the circumferential electrochemically active surface of the working electrode. In addition, a section of the electrochemically active surface of the reference electrode is exposed. For example, the section of the electrochemically active surface can be masked during deposition of the outer insulating layer or etched after deposition of the outer insulating layer.

[0270] In embodiments, the APL is deposited on the substrate containing the aptamer conjugate to obtain a domain thickness of about 0.05 microns or less to about 40 microns or more, more preferably about 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 1, 1.5, 2, 2.5, 3, or 3.5 to about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 microns or more. In one embodiment, the domain thickness of the APL is about 20 microns to about 40 microns, including all ranges and subranges therebetween. In one example, the APL is deposited together with the aptamer conjugate. In one example, the APL (or the APL and aptamer conjugate) is deposited by spray coating or dip coating. The spray process atomizes and mists the solution, allowing most or all of the solvent to evaporate before the coating material settles on the underlying domains, thereby minimizing contact between the solvent and the aptamer. Without wishing to be bound by theory, it is believed that during the process of depositing the APL as described herein, a structural morphology forms around the aptamer that allows for substantially unhindered conformational changes by the aptamer with its target analyte due to the hard-soft multi-segmentation and / or functionalization of the APL structure.

[0271] In embodiments, APL is deposited onto the substrate / coadsorbate by spray coating a solution of about 1% to about 5% by weight polymer and about 95% to about 99% by weight solvent, including all ranges and subranges therebetween. When spraying a solvent-containing solution of APL material onto the substrate / coadsorbate, it is desirable to mitigate or substantially reduce any contact of any solvent in the spray solution with the aptamer, which may inactivate the underlying aptamer, transducer, or redox moiety. As will be appreciated by those skilled in the art, one or more solvents, including water, can be used.

[0272] Although various spraying or deposition techniques can be used, spraying the APL material and rotating the sensor at least once 180 degrees can provide sufficient coverage with APL. Spraying the APL material and rotating the sensor at least twice 120 degrees provides even greater coverage (one layer of 360 degree coverage), thereby ensuring protection against AB or EAB, as described in more detail above.

[0273] In embodiments, the APL is spray- or dip-coated and then cured for about 15 to about 90 minutes at a temperature of about 40 to about 60°C (which can be accomplished under vacuum (e.g., 20 to 30 mmHg)), including all ranges and subranges therebetween. Curing times of up to about 90 minutes or more can be advantageous to ensure complete drying of the APL. While not wishing to be bound by theory, it is believed that complete drying of the APL helps stabilize the sensitivity of the AB or EAB sensor signal. It is believed that complete drying stabilizes the performance of the AB or EAB sensor signal, as it reduces drift in signal sensitivity over time.

[0274] In an embodiment, the APL is formed by spraying or dip-coating one or more layers (e.g., rotating the sensor 120° for 360° coverage) and optionally curing under vacuum at 50°C for 60 minutes. However, the APL can also be formed by dip-coating, depending on the solution concentration, insertion rate, dwell time, withdrawal rate, and / or desired thickness of the resulting APL. In one embodiment, the APL and / or aptamer conjugate is combined with one or more antioxidants. In one embodiment, the antioxidant is incorporated into the aptamer protection layer (APL). In one embodiment, the antioxidant is incorporated into the aptamer protection layer (APL) in an amount of about 0.01% to about 5% by weight. In one embodiment, the antioxidant incorporated into the aptamer protection layer (APL) reduces oxidation of the thiol moiety or thiol-gold bond of the aptamer conjugate, thus increasing the operational and / or shelf life of the EAB of the present disclosure. In one embodiment, the antioxidant is lipophilic, such as vitamin E or other tocopherols. In one embodiment, the antioxidant is butylated hydroxytoluene (BHT). In one embodiment, the antioxidant is added directly to the APL polymer solution (provisional patent filing) and deposited onto the aptamer conjugate using, for example, a multiple dipping process.

[0275] In another example, the antioxidant is hydrophilic, such as ascorbic acid, trehalose, or sodium bisulfite, and is coated as a separate layer or introduced between the aptamer conjugate and the APL. In one example, the antioxidant is grafted onto the APL or to a polymer chain that is miscible or compatible with the APL.

[0276] electronic equipment In one embodiment, the continuous AB or EAB sensor of the present disclosure further comprises one or more of a transmitter, a receiver, a controller, or a power source. Any electronics associated with continuous analyte sensors, such as non-invasive, minimally invasive, and / or invasive (e.g., transcutaneous and fully implantable) sensors, are applicable. For example, sensor electronics and data processing, as well as transceiver electronics, Wi-Fi, Bluetooth, RF, and data processing known in the art, can be incorporated into the AB or EAB sensor of the present disclosure.

[0277] FIG. 14 illustrates an exemplary continuous AB or EAB system 150 configured to measure one or more analytes, alone or in combination with an electrophysiological indicator (e.g., blood pressure, heart rate, core temperature, etc.), as discussed herein. The continuous AB or EAB system 150 includes exemplary continuous AB or EAB devices 100, 200 operably connected to a host 120 and multiple display devices 134a-e, according to certain aspects of the present disclosure. Note that the display device 134e may alternatively or additionally be a drug delivery device capable of acting in cooperation with the continuous AB or EAB system 150 to deliver a drug to the host 120. In one example, the continuous AB or EAB system 150 is an EAB system that is a sensor electronics module 126 and a continuous EAB sensor 122 associated with the sensor electronics module 126. The sensor electronics module 126 may communicate directly and wirelessly with one or more of the multiple display devices 134a-e via wireless communication signals. In one embodiment, the display devices 134a-e may also communicate between each other and / or through each other with the continuous AB or EAB system 150. For ease of reference, wireless communication signals from the analyte sensor system 124 to the display devices 134a-e may be referred to as “uplink” signals 128. For example, wireless communication signals from the display devices 134a-e to the continuous AB or EAB system 150 may be referred to as “downlink” signals 130. Wireless communication signals between two or more of the display devices 134a-e may be referred to as “crosslink” signals 132. Additionally, wireless communication signals may include data transmitted by one or more of the display devices 134a-d to one or more remote servers 140 or network entities, such as cloud-based servers or databases, via “long-range” uplink signals 136 (e.g., cellular signals), and may receive long-range downlink signals 138 transmitted by the remote servers 140.

[0278] The sensor electronics module 126 includes sensor electronics configured to process sensor information and generate converted sensor information. In certain embodiments, the sensor electronics module 126 includes electronic circuitry associated with measuring and processing data from the continuous EAB sensor 122, including predictive algorithms associated with processing and calibrating the continuous analyte sensor data. The sensor electronics module 126 may be integral with (permanently attached to) or removably attached to the continuous EAB sensor 122, achieving a physical connection therebetween. The sensor electronics module 126 may include hardware, firmware, and / or software that enables analyte level measurement. For example, the sensor electronics module 126 may include a potentiostat, a power supply for providing power to the continuous EAB device 122, other components useful for signal processing and data storage, and a telemetry module for transmitting data therefrom to one or more display devices 134a-e. The electronics may be mounted on a printed circuit board (PCB) or the like and may take a variety of forms. For example, the electronics can take the form of an integrated circuit (IC), such as an application-specific integrated circuit (ASIC), an electrochemical analog front end, a microcontroller, and / or a processor. In one embodiment, the electrochemical analog front end comprises a sequencer or waveform synthesizer that creates appropriate waveforms for converting signals from the EAB. Exemplary waveforms include square wave voltammetry, linear sweep voltammetry, cyclic voltammetry, differential pulse voltammetry, AC voltammetry, pulse voltammetry, staircase voltammetry, conventional pulse voltammetry, chronoamperometry, and chronocoulometry.Examples of systems and methods for processing sensor analyte data are described in U.S. Patent Nos. 7,310,544 and 6,931,327, as well as U.S. Patent Application Publication Nos. 2005 / 0043598, 2007 / 0032706, 2007 / 0016381, 2008 / 0033254, 2005 / 0020 3360, U.S. Patent Application Publication No. 2005 / 0154271, U.S. Patent Application Publication No. 2005 / 0192557, U.S. Patent Application Publication No. 2006 / 0222566, U.S. Patent Application Publication No. 2007 / 0203966, and U.S. Patent Application Publication No. 2007 / 0208245, each of which is incorporated herein by reference in its entirety for all purposes.

[0279] Display devices 134a-e are configured to display, alert, and / or justify medication delivery based on sensor information transmitted by sensor electronics module 126 (e.g., in customized data packages transmitted to one or more of display devices 134a-e based on their respective preferences). Each of display devices 134a-e may include a display, such as a touchscreen display, for displaying sensor information to a user (in most cases, host 120 or a caregiver / healthcare professional) and / or receiving input from a user. In some examples, display devices 134a-e may include other types of user interfaces, such as a voice user interface, instead of or in addition to a touchscreen display for communicating sensor information to a user of display device 134a-e and / or accepting user input. In some embodiments, one, some, or all of the display devices 134a-e are configured to display or otherwise communicate the sensor information as it is communicated from the sensor electronics module 126 (e.g., in data packages transmitted to the respective display devices 134a-e) without any additional anticipated processing required for calibration and real-time display of the sensor information.

[0280] 14 , one of the plurality of display devices 134a-e may be a custom display device 134a specifically designed to display a particular type of displayable sensor information (e.g., in some embodiments, numerical values ​​and arrows) associated with the analyte value received from the sensor electronics module 126. In some embodiments, one of the plurality of display devices 134a-e may be a handheld device 134c, such as a mobile phone based on the Android, iOS, or other operating system, a palmtop computer, or the like, which may have a relatively large display and be configured to display a graphical representation of continuous sensor data (e.g., including current and historical data). Other display devices may include a tablet 134d, a smartwatch 134b, a medication delivery device 134e, a blood glucose meter, and / or other handheld devices such as a desktop or laptop computer.

[0281] As mentioned above, because different display devices 134a-e provide different user interfaces, the content of the data package (e.g., the amount, format, and / or type of data to be displayed, alarms, etc.) can be customized (e.g., programmed differently by the manufacturer and / or by the end user) for each particular display device and / or type of display device. Thus, in the example of Figure 14, one or more of the display devices 134a-e can communicate directly or indirectly wirelessly with the sensor electronics module 126 to enable multiple different types and / or levels of display and / or functionality associated with the sensor information, as described in more detail elsewhere herein.

[0282] sterile The APL-EAB of the present disclosure is configured for total or partial sterilization, including aseptic manufacturing and / or packaging. Examples of sterilization methods suitable for the APL-EAB of the present disclosure include, for example, high-energy radiation (UV, e-beam, X-ray), chemical treatment (ethylene oxide, CIDEX OPA™ (0.55% ortho-phthalaldehyde)), or autoclaving.

[0283] Although certain embodiments of the present disclosure have been illustrated with reference to particular combinations of elements, various other combinations may be provided without departing from the teachings of the present disclosure. Thus, the present disclosure should not be construed as being limited to the specific illustrative examples described herein and illustrated in the figures, but may also encompass combinations of elements of the various illustrated embodiments and aspects thereof.

Claims

1. 1. An analyte monitoring sensor configured for in vivo measurement of at least one analyte, comprising: a substrate having a substrate surface; an aptamer protection layer encapsulating at least a portion of the substrate surface, the aptamer protection layer being permeable to the at least one analyte; one or more aptamer conjugates associated with at least a portion of the substrate surface and positioned between the aptamer protection layer and the substrate for obtaining a measurement related to the at least one analyte in vivo; a reversible redox moiety bound to the one or more aptamer conjugates.

2. The analyte monitoring sensor of claim 1 , wherein at least a portion of the substrate is a conductive metal.

3. The analyte monitoring sensor of claim 1 or 2, wherein at least a portion of the substrate is gold, carbon, graphene, or graphene oxide.

4. The analyte monitoring sensor of any one of claims 1 to 3, wherein at least a portion of the substrate comprises pores having an average pore size in nanometer and / or micrometer dimensions.

5. The analyte monitoring sensor of any one of claims 1 to 4, wherein at least a portion of the substrate surface further comprises one or more co-adsorbents.

6. The analyte monitoring sensor of any one of claims 1 to 5, wherein each of the one or more co-adsorbents independently comprises multiple functional groups.

7. The analyte monitoring sensor of any one of claims 1 to 6, wherein the one or more co-adsorbents independently provide one or more of a surface energy range, a pH range, a phase separation range, and an intermolecular interaction range between the one or more aptamers and the aptamer protection layer.

8. The analyte monitoring sensor of any one of claims 1 to 7, wherein the one or more co-adsorbents independently provide ionic strength and are present in an amount capable of adjusting or maintaining the ionic strength in proximity to at least one of the aptamer conjugates.

9. The analyte monitoring sensor of any one of claims 1 to 8, wherein the co-adsorbate comprises a self-assembled monolayer (SAM) associated with the substrate surface.

10. The analyte monitoring sensor of any one of claims 1 to 9, wherein at least a portion of the substrate surface comprises the one or more co-adsorbents and a remaining portion of the substrate surface, and a portion of the remaining portion of the substrate surface comprises the one or more aptamer conjugates.

11. 11. The analyte monitoring sensor of claim 1, wherein at least a portion of the substrate surface comprises the one or more co-adsorbents physically or chemically bound thereto and the remaining portion of the substrate surface comprises the one or more aptamer conjugates physically or chemically bound thereto.

12. 12. The analyte monitoring sensor of claim 1, wherein at least a portion of the substrate surface comprises the one or more co-adsorbents physically or chemically bound thereto, and the one or more aptamer conjugates are physically or chemically bound to the one or more co-adsorbents.

13. The analyte monitoring sensor of any one of claims 1 to 12, wherein the co-adsorbent comprises a mono- or polyfunctional alkanethiol, mercaptoalkanol, alkylmercaptoalkanol, or arylmercaptoalkanol.

14. The analyte monitoring sensor of any one of claims 1 to 13, wherein the thiol functional group of the monofunctional alkanethiol or the polyfunctional alkanethiol is covalently bonded to at least a portion of the substrate surface.

15. The analyte monitoring sensor of any one of claims 1 to 14, wherein the thiol functional group of the monofunctional alkanethiol or the polyfunctional alkanethiol is covalently attached to a gold substrate surface.

16. The analyte monitoring sensor of any one of claims 1 to 15, wherein the co-adsorbent comprises a mono- or poly-functional mercaptoalkanol.

17. The analyte monitoring sensor of any one of claims 1 to 16, wherein the thiol functional group of the monofunctional or polyfunctional mercaptoalkanol is covalently bonded to at least a portion of the substrate surface.

18. The analyte monitoring sensor of any one of claims 1 to 17, wherein the thiol functional group of the monofunctional or polyfunctional mercaptoalkanol is covalently bonded to at least a portion of a gold substrate surface.

19. The analyte monitoring sensor of any one of claims 1 to 18, wherein at least a portion of the substrate surface comprises zwitterionic repeating groups.

20. The analyte monitoring sensor of any one of claims 1 to 19, wherein the zwitterionic repeating group comprises a betaine.

21. The analyte monitoring sensor of any one of claims 1 to 20, wherein the zwitterionic repeating group comprises an ammoniophosphate or a lecithin analog.

22. The analyte monitoring sensor of any one of claims 1 to 21, wherein the zwitterionic repeat group comprises an ammoniophosphonate.

23. The analyte monitoring sensor of any one of claims 1 to 22, wherein the zwitterionic repeating group comprises an ammoniophosphinate.

24. The analyte monitoring sensor of any one of claims 1 to 23, wherein the zwitterionic repeating group comprises ammonio sulfonate.

25. The analyte monitoring sensor of any one of claims 1 to 24, wherein the zwitterionic repeating group comprises ammoniosulfate.

26. The analyte monitoring sensor of any one of claims 1 to 25, wherein the zwitterionic repeating group comprises ammoniocarboxylate.

27. The analyte monitoring sensor of any one of claims 1 to 26, wherein the zwitterionic repeating group comprises an alkanethiol betaine.

28. The analyte monitoring sensor of any one of claims 1 to 27, wherein the alkanethiol is linear and contains multiple betaine groups along the chain.

29. The analyte monitoring sensor of any one of claims 1 to 28, wherein the alkanethiol is a terminally terminated dithiol having at least one betaine group along its chain.

30. The analyte monitoring sensor of any one of claims 1 to 29, wherein the thiol group of the terminally terminated dithiolalkanethiol is covalently attached to the substrate surface.

31. The analyte monitoring sensor of any one of claims 1 to 30, wherein the zwitterionic repeating group comprises a mercaptoalkanol betaine.

32. The analyte monitoring sensor of any one of claims 1 to 31, wherein the mercaptoalkanol is linear and contains multiple betaine groups along the chain.

33. The analyte monitoring sensor of any one of claims 1 to 32, wherein the thiol group of the mercaptoalkanol is covalently attached to the substrate surface.

34. The co-adsorbate bound or tethered to the substrate is represented as follows: 【Chemistry 1】 34. The analyte monitoring sensor of any one of claims 1 to 33, wherein X is -OH, -NHR1, -NH2, or -SH; R1 is branched or unbranched acyclic alkyl, substituted or unsubstituted cyclic alkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heteroalkyl, or substituted or unsubstituted heterocyclic; and a is 1 to 3.

35. The analyte monitoring sensor of any one of claims 1 to 34, wherein at least a portion of the substrate surface comprises a covalently bound amine.

36. The analyte monitoring sensor of any one of claims 1 to 35, wherein at least a portion of the substrate surface comprises a covalently bound aminoalkanoic acid.

37. The analyte monitoring sensor of any one of claims 1 to 36, wherein at least a portion of the carbon, graphene, or graphene oxide substrate surface comprises a covalently bound aminoalkanoic acid.

38. 38. The analyte monitoring sensor of any one of claims 1 to 37, wherein at least a portion of the carbon, graphene, or graphene oxide substrate surface comprises a covalently bound aminoalkanoic acid, which is also covalently bound to the one or more aptamer conjugates.

39. The analyte monitoring sensor of any one of claims 1 to 38, wherein the aptamer protection layer is at least partially cross-linked using an amount of a cross-linking agent.

40. The analyte monitoring sensor of any one of claims 1 to 39, wherein the aptamer protection layer comprises a conductive polymer.

41. The analyte monitoring sensor of any one of claims 1 to 40, wherein the aptamer protecting layer comprises a zwitterionic group.

42. The analyte monitoring sensor of any one of claims 1 to 41, wherein the aptamer protection layer comprises zwitterionic repeating groups.

43. 43. The analyte monitoring sensor of any one of claims 1 to 42, wherein the aptamer protection layer provides an ionic strength or localized pH range, and wherein the zwitterionic repeating group compound is present in an amount capable of adjusting or maintaining the ionic strength or localized pH range.

44. 44. The analyte monitoring sensor of any one of claims 1 to 43, wherein the aptamer protection layer provides a free volume that allows for reversible conformational change of the one or more aptamer conjugates present therein, the free volume being sufficient to provide a signal in the presence of the at least one analyte.

45. The analyte monitoring sensor of any one of claims 1 to 44, wherein the aptamer protection layer comprises a functionalized polymer.

46. The analyte monitoring sensor of any one of claims 1 to 45, wherein the functionalized polymer comprises an alkanethiol group.

47. The analyte monitoring sensor of any one of claims 1 to 46, wherein the alkanethiol group is at the end of the functionalized polymer chain.

48. The analyte monitoring sensor of any one of claims 1 to 47, wherein the alkanethiol groups are present along the backbone of the functionalized polymer chain.

49. The analyte monitoring sensor of any one of claims 1 to 48, wherein the functionalized polymer comprises mercaptoalkanol groups.

50. The analyte monitoring sensor of any one of claims 1 to 49, wherein the mercaptoalkanol group is at the end of the functionalized polymer chain.

51. The analyte monitoring sensor of any one of claims 1 to 50, wherein the mercaptoalkanol groups are present along the backbone of the functionalized polymer chain.

52. The analyte monitoring sensor of any one of claims 1 to 51, wherein the aptamer protection layer comprises a functionalized polymer containing one or more zwitterionic repeat groups.

53. The analyte monitoring sensor of any one of claims 1 to 52, wherein the one or more zwitterionic repeating groups comprises a betaine compound or a derivative thereof.

54. The analyte monitoring sensor of any one of claims 1 to 53, wherein the zwitterionic repeating groups are present at the ends of the functionalized polymer chains.

55. The analyte monitoring sensor of any one of claims 1 to 54, wherein the zwitterionic repeating groups are present along the backbone of the functionalized polymer chain.

56. The analyte monitoring sensor of any one of claims 1 to 55, wherein the functionalized polymer comprises an alkanethiol and a zwitterionic repeating group.

57. The analyte monitoring sensor of any one of claims 1 to 56, wherein the functionalized polymer comprises alkanethiol and betaine groups.

58. The analyte monitoring sensor of any one of claims 1 to 57, wherein the functionalized polymer comprises mercaptoalkanol groups and zwitterionic repeating groups.

59. The analyte monitoring sensor of any one of claims 1 to 58, wherein the functionalized polymer comprises mercaptoalkanol groups and betaine groups.

60. The analyte monitoring sensor of any one of claims 1 to 59, wherein the aptamer protection layer is physically or chemically bound to at least a portion of the substrate surface.

61. 61. The analyte monitoring sensor of any one of claims 1 to 60, wherein the aptamer protection layer is physically or chemically bound to at least a portion of the substrate surface, the one or more aptamer conjugates are physically or chemically bound to at least a portion of the substrate surface, and a substantial remainder of the substrate surface further comprises a physically or chemically bound co-adsorbate.

62. 62. The analyte monitoring sensor of any one of claims 1 to 61, wherein the aptamer protection layer comprises at least one polymer segment selected from the group consisting of polyurethane, polyurea, poly(urethane urea), epoxide, polyolefin, polysiloxane, polyamide, polystyrene, polyacrylate, polyether, polyvinylpyridine, polyvinylpyrrolidone, polyester, polycarbonate, and copolymers thereof.

63. The analyte monitoring sensor of any one of claims 1 to 62, wherein the aptamer protection layer comprises a segmented multi-block polymer.

64. The analyte monitoring sensor of any one of claims 1 to 63, wherein the aptamer protection layer comprises a segmented multi-block polyurethane polymer.

65. The analyte monitoring sensor of any one of claims 1 to 64, wherein the aptamer protection layer comprises a segmented multi-block polyurethaneurea polymer.

66. The analyte monitoring sensor of any one of claims 1 to 65, wherein the segmented multi-block polymer comprises a soft segment and a hard segment.

67. The analyte monitoring sensor of any one of claims 1 to 66, wherein the soft segment is hydrophobic or hydrophilic.

68. The analyte monitoring sensor of any one of claims 1 to 67, wherein the soft segment is hydrophobic and hydrophilic.

69. The analyte monitoring sensor of any one of claims 1 to 68, wherein the soft segment comprises a hydrophobic polyol and a hydrophilic polyol.

70. 70. The analyte monitoring sensor of any one of claims 1-69, wherein the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

71. 71. The analyte monitoring sensor of any one of claims 1 to 70, wherein the soft segment is one or more segments comprising polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymers, and blends or copolymers thereof.

72. The analyte monitoring sensor of any one of claims 1 to 71, wherein the hard segment comprises a urethane group or a urea group.

73. The analyte monitoring sensor of any one of claims 1 to 72, wherein the one or more aptamer conjugates are physically associated with a portion of the substrate surface.

74. The analyte monitoring sensor of any one of claims 1 to 73, wherein the one or more aptamer conjugates are covalently associated with a portion of the substrate surface.

75. The analyte monitoring sensor of any one of claims 1 to 74, wherein the one or more aptamer conjugates comprise an RNA or DNA nucleotide sequence.

76. 76. The analyte monitoring sensor of any one of claims 1 to 75, wherein the one or more aptamer conjugates comprise at least one of a 2'-O-methyl modification of a nucleotide, a disulfide bridge, a 3' cap with an inverted 2-deoxythymidine, a 3'-3'-thymidine linkage at the 3' end, a 2'-F modification, and a double-stranded section.

77. The analyte monitoring sensor of any one of claims 1 to 76, wherein the one or more aptamer conjugates comprise an RNA or DNA sequence having a first linker moiety at the 5' end and the reversible redox moiety at the 3' end, or the one or more aptamer conjugates comprise an RNA or DNA sequence having a first linker moiety at the 3' end and the reversible redox moiety at the 5' end.

78. The analyte monitoring sensor of any one of claims 1 to 77, wherein the first linker moiety on the 5' or 3' end comprises an amino or carboxyl group.

79. The analyte monitoring sensor of any one of claims 1 to 78, wherein the first linker moiety is physically or chemically bound to the substrate at the 5' end or the 3' end.

80. 80. The analyte monitoring sensor of any one of claims 1 to 79, wherein the first linker moiety is physically or chemically attached to the co-adsorbent at the 5' end or the 3' end.

81. The analyte monitoring sensor of any one of claims 1 to 80, wherein the one or more aptamer conjugates are glycopeptide antibiotic-binding aptamers.

82. The analyte monitoring sensor of any one of claims 1 to 81, wherein the one or more aptamer conjugates is a vancomycin-binding aptamer.

83. The analyte monitoring sensor of any one of claims 1 to 82, wherein the one or more aptamer conjugates are neurotransmitter-binding aptamers.

84. 84. The analyte monitoring sensor of any one of claims 1 to 83, wherein the one or more aptamer conjugates are dopamine, L-DOPA, insulin, or glutamate binding aptamers.

85. The analyte monitoring sensor of any one of claims 1 to 84, wherein the one or more aptamer conjugates are carbohydrate, triglyceride, or fatty acid binding aptamers.

86. 86. The analyte monitoring sensor of any one of claims 1 to 85, wherein the one or more aptamer conjugates are glucose, glycerol, or beta-hydroxybutyrate binding aptamers.

87. 87. The analyte monitoring sensor of any one of claims 1 to 86, wherein the one or more aptamer conjugates are physically or chemically bound to a self-assembled monolayer (SAM).

88. 88. The analyte monitoring sensor of any one of claims 1 to 87, wherein the one or more aptamer conjugates are physically or chemically bound to a mono- or poly-functional alkanethiol or mercaptoalkanol.

89. 89. The analyte monitoring sensor of any one of claims 1 to 88, wherein the one or more aptamer conjugates are physically or chemically bound to an alkylthiol betaine.

90. 90. The analyte monitoring sensor of any one of claims 1 to 89, wherein the one or more aptamer conjugates are physically or chemically bound to an aliphatic amine.

91. 91. The analyte monitoring sensor of any one of claims 1 to 90, wherein the one or more aptamer conjugates are physically or chemically bound to an aminoalkanoic acid.

92. The analyte monitoring sensor of any one of claims 1 to 91, wherein the reversible redox moiety comprises iron, iridium, ruthenium, osmium, a thiazine dye, or a derivative thereof.

93. The analyte monitoring sensor of any one of claims 1 to 92, wherein the reversible redox moiety comprises ferrocene or methylene blue.

94. The analyte monitoring sensor of any one of claims 1 to 93, wherein the sensor is configured for continuous, semi-continuous, sequential, or random signal acquisition.

95. The analyte monitoring sensor of any one of claims 1 to 94, wherein the sensor is configured for percutaneous insertion.

96. 96. The analyte monitoring sensor of any one of claims 1 to 95, wherein the sensor further comprises one or more of a reference electrode, a working electrode, and a counter electrode.

97. The analyte monitoring sensor of any one of claims 1 to 96, wherein the sensor further comprises one or more of a transmitter, a receiver, a controller, or a power source.

98. 1. A method for extending the end-of-life of an electrochemical aptamer biosensor (EAB), comprising: electrically associating at least one aptamer conjugate, the at least one aptamer conjugate comprising a reversible redox moiety, with a surface of a conductive substrate; encapsulating the at least one aptamer conjugate in the aptamer protection layer, the at least one aptamer conjugate being configured to undergo a reversible conformational change within the aptamer protection layer in response to interaction with an analyte so as to generate a detectable signal; controlling one or more of the ionic strength within the aptamer protection layer, the pH within the aptamer protection layer, the surface phase separation of the aptamer protection layer, and the intermolecular interaction between the at least one aptamer conjugate and the aptamer protection layer; and extending the end of life of the electrochemical aptamer sensor.

99. 99. The method of claim 98, wherein controlling the ionic strength comprises introducing one or more co-adsorbents into the aptamer protection layer, wherein the one or more co-adsorbents are present in an amount capable of adjusting or maintaining the ionic strength.

100. 100. The method of any one of claims 98-99, wherein the one or more co-adsorbents comprise zwitterionic betaine groups.

101. The zwitterionic betaine group comprises one of the following structures: 【Chemistry 2】 wherein X is —OH, —NHR1, —NH2, or —SH; W, Y, and Z are independently branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, any of which may be optionally substituted with O, OH, halogen, amido, or alkoxyl; and R1 is H, branched or unbranched acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cyclohetero ... aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; R3, R4, and R5 are independently selected from acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, or substituted or unsubstituted heteroaryl; and one or more of R3, R4, R5, W, X, Y, and Z are attached to an aptamer protection layer or a substrate.

102. 102. The method of any one of claims 99 to 101, wherein the co-adsorbent is a terminally terminated dithiol having at least one betaine group along its chain.

103. 103. The method of claim 102, wherein the thiol group of the terminally terminated dithiolalkanethiol is covalently bonded to the substrate surface.

104. 104. The method of any one of claims 99 to 103, wherein the zwitterionic betaine group comprises a mercaptoalkanol betaine or an arylmercaptoalkanol.

105. 105. The method of claim 104, wherein the mercaptoalkanol is linear and contains multiple betaine groups along the chain.

106. 106. The method of any one of claims 105, wherein a thiol group of the mercaptoalkanol or the arylmercaptoalkanol is covalently bound to the substrate surface.

107. 107. The method of any one of claims 98-106, wherein controlling the ionic strength or pH comprises providing one or more zwitterionic betaine groups in the aptamer-protecting layer.

108. 108. The method of any one of claims 98-107, wherein the aptamer protection layer comprises an alkanethiol and one or more zwitterionic groups.

109. 109. The method of any one of claims 98-108, wherein controlling the ionic strength or pH comprises providing the aptamer-protecting layer with mercaptoalkanol groups and zwitterionic betaine groups.

110. 110. The method of any one of claims 98 to 109, wherein controlling the intermolecular interaction between the at least one aptamer conjugate and the aptamer protection layer comprises providing the aptamer protection layer with a segmented multi-block polymer backbone.

111. 111. The method of any one of claims 98 to 110, wherein the segmented multi-block polymer backbone comprises a polyurethane polymer.

112. 112. The method of any one of claims 98-111, wherein the segmented multi-block polymer backbone comprises a polyurethaneurea polymer.

113. 113. The method of any one of claims 98 to 112, wherein the segmented multi-block polymer comprises a soft segment and a hard segment.

114. 114. The method of any one of claims 98 to 113, wherein the soft segment is hydrophobic or hydrophilic.

115. 115. The method of any one of claims 98 to 114, wherein the soft segment is hydrophobic and hydrophilic.

116. 116. The method of any one of claims 98 to 115, wherein the soft segment comprises a hydrophobic polyol and a hydrophilic polyol.

117. 117. The method of any one of claims 98 to 116, wherein the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

118. 118. The method of any one of claims 98-117, wherein the soft segment is one or more segments comprising polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymer, and blends or copolymers thereof.

119. 119. The method of any one of claims 98 to 118, wherein the segmented multi-block polymer comprises a soft segment and a hard segment.

120. 120. The method of any one of claims 98 to 119, wherein the hard segments comprise urethane or urea groups.

121. 121. The method of any one of claims 98 to 120, wherein the soft segment is hydrophobic or hydrophilic.

122. 122. The method of any one of claims 98 to 121, wherein the soft segment is hydrophobic and hydrophilic.

123. 123. The method of any one of claims 98 to 122, wherein the soft segment comprises a hydrophobic polyol and a hydrophilic polyol.

124. 124. The method of any one of claims 98 to 123, wherein the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

125. 125. The method of any one of claims 98-124, wherein the soft segment is one or more segments comprising polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymer, and blends or copolymers thereof.

126. 126. The method of claim 98, wherein reducing biofouling comprises providing an aptamer protection layer according to any one of claims 98-124.

127. 127. The method of any one of claims 98 to 126, wherein reducing the separation of the at least one aptamer from the surface of the conductive substrate comprises binding the at least one aptamer conjugate to the conductive surface.

128. 128. The method of any one of claims 98-127, wherein reducing oxidation of the aptamer comprises incorporating one or more non-diffusible antioxidants into the aptamer protection layer.

129. 129. The method of any one of claims 98-128, wherein controlling the diffusion of the at least one aptamer comprises at least partially cross-linking the aptamer protection layer.

130. 130. The method of any one of claims 98 to 129, wherein end of life is extended by up to 1 day, 2 days, 1 week, 2 weeks, 3 weeks, or 1 month.

131. 1. An aptamer protection layer configured for transcutaneous in vivo continuous online monitoring, comprising: a functionalized polymer comprising at least one zwitterionic repeating group; Polymerizable zwitterionic monomer structures such as: 【Transformation 3】 (wherein X is O, NH, or NR ４ wherein Y and Z are independently acyclic alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloheteroalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, which may optionally be substituted with OH, halogen, or alkoxyl; and R １ , R ３ , R ４ , and R ５ is independently H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; functionalized polymers containing alkanethiol, phenylthiol, or benzylthiol groups; functionalized polymers comprising alkanethiol, phenylthiol, or benzylthiol groups in combination with zwitterionic repeating groups; functionalized polymers comprising mercaptoalkanol groups, arylmercaptoalkanol groups, benzylmercaptoalkanol groups, or mixtures thereof; a functionalized polymer comprising mercaptoalkanol groups, arylmercaptoalkanol groups, benzylmercaptoalkanol groups, or mixtures thereof in combination with zwitterionic repeating groups; or An aptamer protection layer comprising a polymer selected from segmented multi-block polymers.

132. The aptamer protection layer of claim 131, wherein the aptamer protection layer is at least partially crosslinked.

133. The aptamer protection layer of any one of claims 131 to 132, wherein the aptamer protection layer provides ionic strength and the zwitterionic repeat group compound is present in an amount capable of adjusting or maintaining ionic strength or pH in proximity to the aptamer conjugate.

134. 134. The aptamer protection layer of any one of claims 131 to 133, wherein the aptamer protection layer provides a free volume that allows for a reversible conformational change of one or more of the aptamer conjugates present sufficient to provide a detectable signal in the presence of an analyte.

135. The aptamer protection layer of any one of claims 131 to 134, wherein the alkanethiol group is present at the end of the functionalized polymer chain.

136. The aptamer protection layer of any one of claims 131 to 135, wherein the alkanethiol groups are present along the backbone of the functionalized polymer chain.

137. The aptamer protection layer of any one of claims 131 to 136, wherein the mercaptoalkanol group is present at the end of the functionalized polymer chain.

138. The aptamer protection layer of any one of claims 131 to 137, wherein the mercaptoalkanol groups are present along the backbone of the functionalized polymer chain.

139. The aptamer protection layer of any one of claims 131 to 138, wherein the zwitterionic repeating groups are present at the ends of the functionalized polymer chains.

140. The aptamer protection layer of any one of claims 131 to 139, wherein the zwitterionic repeating groups are present along the backbone of the functionalized polymer chain.

141. The aptamer protection layer of any one of claims 131 to 140, wherein one or more of the zwitterionic repeat groups comprises a betaine compound or a derivative thereof.

142. The aptamer protection layer of any one of claims 131 to 141, wherein the aptamer protection layer is configured to be physically or chemically bound to at least a portion of the substrate surface.

143. The aptamer protection layer of any one of claims 131 to 142, wherein the segmented multiblock polymer comprises at least one of polyurethane, polyurea, poly(urethane urea), epoxide, polyolefin, polysiloxane, polyamide, polystyrene, polyacrylate, polyether, polyol, polyvinylpyridine, polyvinylpyrrolidone, polyester, polycarbonate, and copolymers thereof.

144. The aptamer protection layer of any one of claims 131 to 143, wherein the aptamer protection layer comprises a segmented multi-block polyurethane polymer.

145. The aptamer protection layer of any one of claims 131 to 144, wherein the aptamer protection layer comprises a segmented multi-block polyurethane urea polymer.

146. The aptamer protection layer of any one of claims 131 to 145, wherein the segmented multiblock polymer comprises a soft segment and a hard segment.

147. The aptamer protection layer described in any one of claims 131 to 146, wherein the soft segment is hydrophobic or hydrophilic.

148. The aptamer protection layer of any one of claims 131 to 147, wherein the soft segment is hydrophobic and hydrophilic.

149. The aptamer protection layer of any one of claims 131 to 148, wherein the soft segment comprises a hydrophobic polyol and a hydrophilic polyol.

150. The aptamer protection layer of any one of claims 131 to 149, wherein the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

151. The aptamer protection layer of any one of claims 131 to 150, wherein the soft segment is one or more segments comprising polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymer, and blends or copolymers thereof.

152. The aptamer protection layer of any one of claims 131 to 151, wherein the aptamer protection layer has an average molecular weight of about 10 kDa to about 500 kDa.

153. 1. A method for determining the in vivo concentration of an analyte, comprising: contacting a biological fluid containing an analyte in vivo with an electrochemical aptamer biosensor bound to a conductive substrate, wherein an aptamer probe is encapsulated in an aptamer protection layer, the aptamer protection layer being permeable to the analyte, and the electrochemical aptamer biosensor generates a signal upon interaction with the analyte; interrogating the conductive substrate or the electrochemical aptamer; detecting said signal corresponding to an in vivo concentration of said analyte.

154. 154. The method of claim 153, wherein said investigating is continuous, semi-continuous, sequential, or random detection of said signal.

155. 154. The method of claim 153, further comprising adjusting the signal based on a background signal generated as a result of non-specific binding of the aptamer biosensor to generate an adjusted signal.

156. 156. The method of any one of claims 153 to 155, further comprising determining the in vivo concentration of the analyte over a period of time based on the adjusted signal.

157. 157. The method of any one of claims 153 to 156, wherein investigating the conductive substrate comprises a differential measurement technique.

158. 158. The method of claim 157, wherein the differential measurement technique includes investigating the conductive substrate at a first square wave voltammetry (SWV) frequency to obtain a first signal and at a second SWV frequency to obtain a second signal, taking the difference between the two signals, and dividing by the average of the two signals to obtain an adjusted signal.

159. 157. The method of any one of claims 153 to 156, wherein the investigating comprises chronoamperometry.

160. 157. The method of any one of claims 153 to 156, wherein the investigating comprises cyclic voltammetry.

161. 161. The method of any one of claims 153 to 160, wherein the conductive substrate is an electrode, a microporous, or a nanoporous conductive material.

162. 1. A method for manufacturing an electrochemical aptamer biosensor (EAB), comprising: presenting at least one aptamer, wherein at least one aptamer conjugate comprises a reversible redox moiety, on at least a portion of a surface of a conductive substrate; presenting an aptamer protection layer on a portion of the surface of the conductive substrate; and encapsulating at least a portion of said at least one aptamer conjugate in said aptamer-protecting layer.

163. 163. The method of claim 162, further comprising including one or more co-adsorbents in the aptamer protection layer.

164. 164. The method of claim 162 or 163, wherein the one or more co-adsorbents comprise a zwitterionic betaine group.

165. the one or more zwitterionic betaine groups are selected from the following structures: 【Chemistry 4】 wherein W, Y, and Z are independently branched or straight chain alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, any of which may be optionally substituted with O, OH, halogen, amido, or alkoxyl; R １ is H, alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl; R ２ , R ３ , and R ４ is independently selected from alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl, and one or more of R, R, R, W, X, Y, and Z are bound to the co-adsorbent.

166. 166. The method of any one of claims 162 to 165, wherein the co-adsorbate is a terminally terminated dithiol having at least one betaine group along its chain.

167. 167. The method of claim 166, wherein the thiol group of the terminally terminated dithiolalkanethiol is covalently bonded to the substrate surface.

168. 168. The method of any one of claims 162 to 167, wherein the zwitterionic betaine group comprises a mercaptoalkanol betaine.

169. 169. The method of claim 168, wherein the mercaptoalkanol is linear and contains multiple betaine groups along the chain.

170. 170. The method of any one of claims 162 to 169, wherein the thiol group of the mercaptoalkanol is covalently attached to the substrate surface.

171. 171. The method of any one of claims 162-170, wherein the aptamer protection layer comprises an alkanethiol and one or more zwitterionic betaine groups.

172. 172. The method of any one of claims 162-171, wherein the aptamer protection layer comprises a segmented multi-block polymer backbone.

173. 173. The method of any one of claims 162-172, wherein the segmented multi-block polymer backbone comprises a polyurethane polymer.

174. 174. The method of any one of claims 162-173, wherein the segmented multi-block polymer backbone comprises a polyurethaneurea polymer.

175. 175. The method of any one of claims 162 to 174, wherein the segmented multi-block polymer comprises a soft segment and a hard segment.

176. 176. The method of any one of claims 162 to 175, wherein the soft segment is hydrophobic or hydrophilic.

177. 177. The method of any one of claims 162 to 176, wherein the soft segment is hydrophobic and hydrophilic.

178. 178. The method of any one of claims 162 to 177, wherein the soft segment comprises a hydrophobic polyol and a hydrophilic polyol.

179. 179. The method of any one of claims 162 to 178, wherein the soft segment is one or more segments comprising polydimethylsiloxane, polycarbonate, polyester, polyether, and blends or copolymers thereof.

180. 180. The method of any one of claims 162-179, wherein the soft segment is one or more segments comprising polyethylene glycol, oligopolyether, polyoxazoline (POX), polypeptide, polyvinylpyrrolidone, polyvinylpyridine, zwitterionic repeating group polymer, and blends or copolymers thereof.

181. 181. The method of any one of claims 162 to 180, wherein the segmented multi-block polymer comprises a soft segment and a hard segment.

182. 182. The method of any one of claims 162 to 181, wherein the hard segments comprise urethane or urea groups.

183. 183. The method of any one of claims 162 to 182, wherein the aptamer protection layer is cross-linked using an amount of a cross-linking agent.

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